CA2340586A1 - T-type calcium channel - Google Patents

Abstract

The present invention is directed to isolated nucleic acid molecules encoding pancreatic T-type calcium channels and vectors and host cells comprising such.
The invention is further directed to methods and compositions which modulate the expression of pancreatic T-type calcium channels, including antisense. An isolated pancreatic T-type calcium channel protein is provided, as well as antibodies directed to such protein. Pharmaceutical compositions and methods of treatment involving pancreatic T-type calcium channels are also provided.

Description

T-TYPE CALCIUM CHANNEL
This application claims priority of U.S. Provisional Patent Application No. 60/098,004, filed August 26, 1998;
5 and of U.S. Provisional Patent Application No.
60/117,399, filed January 27, 1999. .-The subject matter of this application was made with support from the United States Government under National Institutes of Health Grant No. 5-20174 . The U.S.
Government may have certain rights in this invention.
FIELD OF THE INVENTION
The present invention relates generally to calcium 15 channel proteins, and more particularly to pancreatic T-type calcium channel proteins and uses thereof.
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
25 Insulin secretion from pancreatic [3-cells is the primary physiological mechanism c>f blood glucose regulation. A rise in blood glucose concentration stimulates release of insulin from the pancreas, which in turn promotes glucose uptake in peripheral tissues and 30 consequently lowers blood glucose levels, reestablishing euglycemia. Non-insulin dependent diabetes mellitus (NIDDM)(type II diabetes) is associated with an impairment in glucose-induced insulin secretion in pancreatic ~i-cells (Vague and Moulin, 1982) 35 Voltage-gated Ca2+ channels mediate a rapidly activated inward movement of Ca2* ions that underlies the WO 00/15845 PCTlUS99/I9175 stimulation of insulin secretion in ~i-cells (Boyd III
1991) . In different tissues, four types of Ca2* channels have been described (L(P/Q), T, N,, and E channels). The purified L-type Caz* channel consists of five subunits: al, 5 a2, (3, Y, b (Catterall 1991) . The primary structure of the al subunit is organized in four homologous domains containing six transmembrane segments (Catterall 1988).
Rat arid human pancreatic (3-cells are equipped with L-type and T-type Ca2* channels (H:iriart and Matteson, 1988; Davalli et al . , 1996) . L-t~Tpe Ca2* channels, activated at high voltages and having large unitary conductance and dihydropyridine-sensitivity, are considered the major pipeline for Ca2* influx into the (3-cell (Keahey et al., 1989). In contrast, T-type calcium 15 channels activate at low voltages and have small unitary conductance and dihydropyridine-insensitivity.
The physiological function oi= T-type Caz* channels in [i-cell insulin-secretion has been demonstrated (Bhattacharjee et al., 1997). Thf~se channels facilitate 20 exocytosis by enhancing electrica:L activity in these cells. L-type and T-type Caz* channels, under normal conditions, work in concert promoting the rise in [Ca2*)i during glucose-stimulated insulin secretion. In (3-cells, over-expressed T-type Caz+ channels may be, at least in 25 part, responsible for the hyper-rE=_sponsiveness of insulin secretion to non-glucose depolari:aing stimuli in GK rat and in rat with NIDDM induced by neonatal injection of streptozotocin (Kato et al., 1994; Kato et al., 1996).
However, over-expressed T-type ca:Lcium channels over time 30 will ultimately lead to an elevation of basal Caz* through it's window current properties. 'therefore, there is a dual effect of T-type Caz* channels in ~-cells depending upon channel number and membrane potential.

_ 3 - _ Two isoforms of L-type Ca2+ channel al subunits have been identified in (3-cells (Seino et al., 1992; Yaney et al., 1992). The rat neuronal T-type calcium channel has recently been cloned (Perez-Reye~s et al., 1998). Other subunits of T-type Ca2' channel have yet to be identified.-Given the evidence that T-type calcium channels are associated with type II diabetes" a need exists to further characterize T-type calcium channels.
SUMMARY OF THE hNVENTION
To this end, the subject invention provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel. The invention also provides an antisense nucleic acid molecule complementary to at least a portion of the mRNA encoding th.e pancreatic T-type calcium channel.
The, isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the pancreatic T-type calcium channel results in production of pancreatic T-type calcium channel in a host cell. Expression of the antisense nucleic acid molecules in a host cell results in decreased expression of the pancreatic T-type calcium channel.
The invention further provides a ribozyme having a recognition sequence complementary to a portion of mRNA
encoding a pancreatic T-type calcium channel. The ribozyme can be introduced into a cell to also achieve decreased expression of pancreatic: T-type calcium channel in the cell.
The invention further provides a method of screening a substance for the ability of the substance to modify T-type calcium channel function, and. a method of obtaining DNA encoding a pancreatic T-type calcium channel.

WO 00/15845 PCT/US99I19~75 Further provided is an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel, wherein the nucleic acid molecule encodes a first amino acid sequence having at least 90o amino acid identity to 5 a second amino acid sequence. Th.e second amino acid sequence is as shown in SEQ ID N0:2.
The invention further provides a DNA oligamer capable of hybridizing to a nucleic acid molecule encoding a pancreatic T-type calcium channel. The DNA
10 oligomer can be used in a method of detecting presence of a pancreatic T-type calcium channel in a sample, which method is also provided by the subject invention.
The invention also provides an isolated pancreatic T-type calcium channel protein, a:nd antibodies or 15 antibody fragments specific for t:he pancreatic T-type calcium channel protein. The antibodies and antibody fragments can be used to detect the presence of the pancreatic T-type calcium channel protein in samples.
Further provided is an isolated pancreatic T-type calcium 20 channel protein encoded by a first amino acid sequence having at least 90%: amino acid idc=ntity to a second amino acid sequence, the second amino acid sequence as shown in SEQ ID N0:2.
The subject invention further provides a method of 25 modifying insulin secretion by pancreatic beta cells, the method comprising modifying level: of functional T type calcium channels in the pancreatic: beta cells. The invention further provides a method of treating type II
diabetes in a subject, the method comprising 30 administering to the subject an amount of a compound effective to modify levels of functional T type calcium channel in the pancreatic beta cells of the -subject.
The invention also provides a method of modifying basal calcium levels in cells, a method of modifying the WO 00/15845 PCTlUS99/?9675 action potential of L type calcium channels in cells, a method of modifying pancreatic beta cell death, a method of modifying pancreatic beta cell proliferation, and a method of modifying calcium influ:K through L type calcium 5 channels in cells, each of the methods comprising modifying levels of functional T ~~ype calcium channels in the cells.
BRIEF DESCRIPTION OF THE DRAWINGS
10 These and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which:
Fig. lA illustrates a comparison of the nucleotide 15 sequence of a1G-INS (1) and a1G (2;) at the 5~-end regions (aal-67 of a1G). The tour insertions are indicated with arrow heads. The capital ATG represents the start codon far each cDNA;
Fig. 1B is a schematic illustration representing 20 partial rat genomic nucleotide composition between Domain III and IV. Genomic DNA contained an exon specific to a1G
(shaded circle) and an exon specific to the al subunit of T-type Ca2' deduced from INS-1 (shaded rectangle) between 4845 and 5256 of the cDNA sequence'. Other exons (open 25 rectangles) are identical between the two cDNAs. The bold letters indicate the nucleotides coding Gly-1667;
Figs. 2A-2D illustrate expre~~sion of a1G-INS in Xenopus oocytes. Fig. 2A illustrates 40 mM Caz+ currents elicited by depolarizing pulses from - 60 to 40 mV. Fig.
30 2B illustrates time constants of activation and inactivation measured at test potentials between -30 and 30 mV. The time constants of activation were obtained by fitting the increasing portion (activation) of currents with the Hodgkin-Huxley equation where the m value was WO 00/15845 PCT/US99l19675 designated as four (n = 6). The time constants of inactivation were obtained by single exponential fitting (n = 6). Fig. 2C illustrates voltage-dependent conductance (n = 7) and Fig. 2D illustrates steady-state inactivation (n = 3) of expressed currents in oocytes.
The holding potential for Figs. 2C and 2D was -80 mV. The currents in Fig. 2D were measured at -10 mV after varying 1000 ms pre-pulse potentials. Peak currents were normalized to the maximum current and then averaged (error bars represent SE);
Figs. 3A and 3B illustrate a~~cumulative dose response relationships of the inhibitory effects of mibefradil on T- and L-type Caz+ currents. Currents were measured with the whole--cell patch clamp configuration.
Data from four experiments were normalized individually and than plotted as mean ~ standard error. Fig. 3A
illustrates curve which was generated by fitting the data using one-to-one binding curve according to the equation 1/ (1 + [mibefradil] /Kd) . Fig. 3B is a dose response of L-type Ca2+ current obtained when perfusion of solutions containing different concentrations of mibefradil;
Fig. 4 illustrates reversibility of the inhibition of T and L-type currents by NiClz and mibefradil, respectively. Open and solid circles represent the T-type Ca2+ current recorded before and after NiClz (2 ~.1 of 30 ~.M) and mibefradil (2 ~,1 of 10 ~.M; were administrated, respectively. The open squares represent the L-type Ca2+
current recorded before and after mibefradil (2 ~cl of 10 ~M) was administrated with perforated patch clamp configuration. The T-type Ca2' current was measured at -30 mV with a holding potential of -8t) mV with whole cell configuration. Arrow indicates thE: time when the drugs were delivered. n =3 for each group experiments;

- 7 _ Figs. SA and 5B illustrate the long-term effect of mibefradil (10 nM) on L- and T- Ca2+ currents in the perforated-patch configuration. In Fig. 5A, solid and open circles represent the L-type Caz' current recorded in the cells with and without administration of mibefradil, -respectively. Solid triangles represent T-type Ca2' currents recorded in the cells after administrating mibefradil. Mibefradil were delivered at time zero. n = 4 for each group experiments. In F:ig. 5B, cells were cultured in medium with or withoul~ co-incubating 10 nM
mibefradil for 2 hours. The current densities were recorded with perforated patch c1<~mp configuration. n =
14 for each group experiments;
Fig. 6A illustrates accumulation of dm-mibefradil in the cells measured with mass spectrometry. The cells were first incubated with mibefradil (:?0 ~M) for the duration indicated on the figure (n = 3). The inset (Fig. 6B) shows the primary data of mass spectrometry indicating peaks at 496 and 424, which corre:~pond to mibefradil and dm-mibefradil, respectively;
Fig. 7A illustrates the effect of mibefradil and dm-mibefradil on L-type Caz+ currents from inside cells. n - 8, *, p < 0.01 to the control;
Fig. 7B illustrates the effects of mibefradil or dm-mibefradil on T-type Caz' current from inside cells n =
4. All data were collected at 5 min after formation of whole cell patch. The pipette solution contained 1 ~.M of drug;
Fig. 8 illustrates basal [Ca2+]i measured in an INS-1 cell. T-type calcium channel antagonist mibefradil (1 ACM) reduced basal [Ca2'] i in a single cell in the bath solution without glucose. The [Ca2+]i was measured with the emission ratio of Fura-2 AM (F'380/F340) then WO 00/15845 PCT/US99/t9b75 _ 8 _ _ _ calibrated with the standard solution purchased from Molecular Probes Inc. (OR);
Fig. 9A illustrates that intracellular perfusion of a solution containing 272 nM free calcium concentration inhibits the L-type calcium current. Currents were -elicited by a step voltage to +10 mV, with holding potential of -80 mV;
Fig. 9B illustrates the effects of perfusing in high calcium concentration on the IV calcium current relationship. Closed circles represent the cell before perfusion, and open circles repre~~ent perfusion of 272 nM
free calcium;
Fig. 9C illustrates the effect of intracellular perfusion of different calcium concentrations on L-type calcium current over time. Squares represent perfusion from high calcium to low calcium (intracellular solution contained 632 nM then perfused by a solution with 10 mM
EGTA), triangles represent perfusion from low calcium to 272 nM calcium, and circles represent low calcium to 632 nM calcium;
Fig. 9D illustrates the effect of high calcium on the T-type calcium channel current. Tail currents were elicited by a voltage step to -30 mV for 10 ms;
Fig. 10 illustrates that reestablishment of basal calcium causes stereotyped calcium influx. A cell was twice perfused with 50 mM KC1 with an intervening perfusion of the original bath solution to restore membrane potential;
Fig. 11 illustrates that elevated basal Ca2+ causes a defect in the Cazi transient. A cell was twice perfused with 50 mM KC1 with an intervening perfusion of the original bath solution to restore membrane potential.
The second perfusion occurred prior to reestablishment of the original basal [Ca2') i of about 60 nM;

- 9 _ _ Fig. 12 illustrates a model for glucose-stimulated insulin release;
Fig. 13 illustrates that mibefradil (1 ~M) blocks T-and L-type Ca2+ current in INS-1 cells. The relative 5 current of T type Ca channel is obtained by measuring -their slow deactivated tail current (n = 8);
Fig. 14 illustrates that mibefradil and NiCl2 reversibly block T type Ca2* current in INS-1 cells.
Drugs were administered into the recording chamber at 180 seconds from the beginning of recording. N = 3;
Fig. 15 illustrates the activation and inactivation curves for INS-1 cells, revealing a "window current";
Fig. 16 illustrates the effect of NiCl2, mibefradil, and nifedipine on basal insulin secretion in NIT-1 cells.
The glucose concentration is 3 mM in the experiments;
Fig. 17 illustrates that the T type calcium channel antagonist NiClz (30 ACM) reduced t:he frequency of transient spontaneous elevation of [Ca2*]i, in a single cell in the bath solution without glucose;
20 Fig. 18 illustrates the effect of 30 mM NiCl2 on the [Ca2+]i under non-stimulus conditions. Data was collected from the cells with "high" initial basal [Caz*]i (about 100 nM). n = 13;
Figs. 19A and 19B illustrate that hyperpolarization 25 induced an increase in number of i~ction potentials and a decrease in onset latencies. N = 40;
Figs. 20A and 20B illustrate the dose-dependent effect of NiCl~ on insulin secretion. Cells were placed in a medium containing 11.1 mM glucose and a decrease in 30 onset latencies. N = 40;
Fig. 21 illustrates "run-up" in whole cell recording;
Fig. 22 illustrates KCl induced Ca2* influx in the INS-1 cells treated with streptozotocin. n = 13;

Fig. 23A-23F illustrate the results of cytokine treatment. LVA Ca2* currents were induced by cytokine treatment (IL-1(3, 25 U/ml; IFNy, 300 U/ml} for 6 h in primary cultured mouse islet cells, but not in a-TC1 5 cells. An LVA current was elicited by a -40 mV test -pulse in an islet cell (Fig. 23A), but the same current was not detected in a-TC1 cells (Fig. 23C}. The Ca2*
current density-voltage relationships obtained from islet cells (Fig. 23B) and a-TCl cells (Fig. 23D) with and 10 without cytokine treatment are shown. The open circles represent the current densities of untreated cells (n =
for islet cells; n = 20 for a-TC1 cells), and the filled circles represent the current densities of cells treated by cytokines (n = 21 for islet cells; n = 21 for 15 a-TC1 cells). The recordings were elicited by voltages ranging from -50 to +20 mV for 100 msec. All experiments were performed at -80 mV. Fig. 23E shows steady state inactivation of LVA tail currents elicited by a 10-msec depolarizing (-10 mV) pulse followed by a 50-msec 20 hyperpolarizing pulse (-100 mV), with a holding patential of -80 mV. Fig. 23F shows that NiCl2 (10 ~.M) blocked the cytokine induced LVA Ca2* current elicited at a -30 mV
step pulse in an islet cell;
Figs. 24A and 24B illustrate the effects of 25 cytokines on [Ca2*]i in mouse islet cells and a-TC1 cells.
In Fig. 24A, basal [Caz*] i of primary cultured mouse islet cells was approximately 3-fold higher after cytokine treatment. NiCl2 (10 ~.M) , but not; nifedipine (10 ACM) , prevented the increase in [Ca2*]i. In Fig. 24B, basal 30 [Ca2*]i in a-TC1 cells was unaffected by cytokine treatment. Cytokine treatment consisted of IL-1[i (25 U/ml) and IFNy (300 U/ml) for 6 h; and Figs. 25A and 25B illustrate the effects of Ni.Cl2 on cytokine-induced a-TC3 cell death. NiCl2 (20 ~M) significantly reduced cell death induced by cytokines in both a time (Fig. 25A).and dose-dependent {Fig. 25B) manner (n = 3). Cytokine treatment consisted of IL-1(3 {25 U/ml), IFNy {100 U/ml), and TNFa (100 U/ml) in Fig.
5 25A and of IL-1(3 {25 U/ml), TNFa (100 U/ml), and various -concentrations of IFNy as indicated in Fig. 25A. The first dose, 0, represents zero concentration for all three cytokines. The concentration of nifedipine was 10 ~M in both Fig. 25A and Fig. 25B.
to DETAILED DESCRIPTION OF' THE INVENTION
The term "nucleic acid", as used herein, refers to either DNA or RNA. "Nucleic acid sequence"' or "polynucleotide sequence" refers to a single- or 15 double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA, and nonfunctional DNA or RNA.
"Isolated" nucleic acid refers to nucleic acid which 20 has been separated from an organism in a substantially purified form {i.e. substantially free of other substances originating from that organism), and to synthetic nucleic acid.
By a nucleic acid sequence "homologous to" or 25 "complementary to", it is meant a nucleic acid that selectively hybridizes, duplexes or binds to DNA
sequences encoding the protein (channel) or portions thereof when the DNA sequences encoding the protein are present in a human genomic or cDNA library. A DNA
30 sequence which is similar or complementary to a target sequence can include sequences wh:i.ch are shorter or longer than the target sequence so long as they meet the functional test set forth.

- 12 - ' Typically, the hybridization is done in a Southern blot protocol using a 0.2X SSC, O.lo SDS, 65"C wash. The term "SSC" refers to a citrate-saline solution of 0.15M
sodium chloride and 20 mM sodium citrate. Solutions are often expressed as multiples or fractions of this -concentration. For example, 6X S;SC refers to a solution having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate. 0.2X SSC refers to a solution 0.2 10 times the SSC concentration or 0.03M sodium chloride and 4 mM sodium citrate.
The phrase "nucleic acid molecule encoding" refers to a nucleic acid molecule which directs the expression of a specific protein or peptide. The nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA aequence that is translated into protein or peptide. The nucleic acid molecule includes both the full length nucleic acid sequences as well as non-full length sequences derived 20 from the full length protein. It being further understood that the sequence includes the degenerate codons of the native sequence or :sequences which may be introduced to provide codon preference in a specific host cell.
25 The term "located upstream" ~~s used herein refers to linkage of a promoter upstream from a nucleic acid (DNA) sequence such that the promoter mediates transcription of the nucleic acid (DNA) sequence.
The term "vectors', refers to viral expression 30 systems, autonomous self-replicat~Lng circular DNA
(plasmids), and includes both expression and nonexpression plasmids. Where a recombinant microorganism or cell is described as hosting an "expression vector," this include~~.both extrachromosomal x3 circular DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is, being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous 5 structure, or the vector may be incorporated within.the -host's genome.
The term "plasmid" refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both the expression and nonexpression types.
10 Where a recombinant microorganism or cell is described as hosting an "expression plasmid", this includes latent viral DNA integrated into the host chromosome(s). Where a plasmid is being maintained by a host cell, the plasmid is either being stably replicated by the cell during 15 mitosis as an autonomous structure, or the plasmid is incorporated within the host's genome.
The phrase "heterologous protein" or "recombinantly produced heterologous protein" refers to a peptide or protein of interest produced using cells that do not have 20 an endogenous copy of DNA able to express the peptide or protein of interest. The cells produce the peptide or protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequences.
The recombinant peptide or protein will not be found in 25 association with peptides or proteins and other subcellular components normally associated with the cells producing the peptide or protein.
The following terms are used to describe the sequence relationships between two or mare nucleic acid 30 molecules or polynucleotides, or between two or more amino acid sequences of peptides or proteins: "reference sequence", "comparison window", "sequence identity"
"sequence homology", "percentage of sequence identity", "percentage of sequence homology", "substantial i, identity", and "substantial homology". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a 5 full-length cDNA or gene sequence: given in a sequence -listing or may comprise a complete cDNA or gene sequence.
Optimal alignment of sequences for aligning a comparison window may be conducted, for example, by the local homology algorithm of Smith. and Waterman (1981), by l0 the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics 15 Computer Group, 575 Science Dr., Madison, Wis.).
As applied to nucleic acid molecules or polynucleotides, the terms "substantial identity" or "substantial sequence identity" mean that two nucleic acid sequences, when optimally aligned (see above), share 20 at least 90 percent sequence identity, preferably at least 95 percent sequence identity, more preferably at least 96, 97, 98 or 99 percent sequence identity.
"Percentage nucleotide (or nucleic acid) identity"
or "percentage nucleotide (or nucleic acid) sequence 25 identity" refers to a comparison of the nucleotides of two nucleic acid molecules which, when optimally aligned, have approximately the designated percentage of the same nucleotides. For example, "95% nucleotide identity"
refers to a comparison of the nucleotides of two nucleic 30 acid molecules which when optimally aligned have 950 nucleotide identity. Preferably, nucleotide positions which are not identical differ by redundant nucleotide substitutions (the nucleotide substitution does not change the amino acid encoded by the particular codan).

WO 00/15$45 PCT/US99/19675 _ 15 _ ..
As further applied to nucleic acid molecules or polynucleotides, the terms "substantial homology" or "substantial sequence homology" mean that two nucleic acid sequences, when optimally aligned (see above), share 5 at least 90 percent sequence homology, preferably at -least 95 percent sequence homology, more preferably at least 96, 97, 98 or 99 percent sequence homology.
"Percentage nucleotide (or nucleic acid) homology"
or "percentage nucleotide (or nucleic acid} sequence 10 homology" refers to a comparison of the nucleotides of two nucleic acid molecules which, when optimally aligned, have approximately the designated percentage of the same nucleotides or nucleotides which .are not identical but differ by redundant nucleotide substitutions (the 15 nucleotide substitution does not change the amino acid encoded by the particular codon}. For example, "95%
nucleotide homology" refers to acomparison of the nucleotides of two nucleic acid molecules which when optimally aligned have 95% nucleotide homology.
20 As applied to polypeptides, 'the terms "substantial identity" or "substantial sequence identity" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFTT using default gap, share at least 90 percent sequence identit~~, preferably at least 25 95 percent sequence identity, more preferably at least 96, 97, 98 or 99 percent sequence identity.
"Percentage amino acid identity" or "percentage amino acid sequence identity" refers to a comparison of the amino acids of two polypeptidc=s which, when optimally 30 aligned, have approximately the dE=_signated percentage of the same amino acids. For examplea, "95% amino acid identity" refers to a comparison of the amino acids of two polypeptides which when optimally aligned have 95%
amino acid identity. Preferably, residue positions which WO 00/15845 PCT/US99/~ 9b75 - 16 - _ are not identical differ by conservative amino acid substitutions. For example, the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to affect the properties of a 5 protein. Examples include glutamine for asparagine or -glutamic acid for aspartic acid.
As further applied to polype;ptides, the terms "substantial homology" or "substantial sequence homology"
mean that two peptide sequences, when optimally aligned, 10 such as by the programs GAP or BE;STFIT using default gap, share at least 90 percent sequence homology, preferably at least 95 percent sequence homology, more preferably at least 96, 97, 98 or 99 percent sequence homology.
"Percentage amino acid homology" or "percentage 15 amino acid sequence homology" refers to a comparison of the amino acids of two polypeptid.es which, when optimally aligned, have approximately the designated percentage of the same amino acids or conservatively substituted amino acids. For example, "95°s amino acid homology" refers to 20 a comparison of the amino acids of two polypeptides which when optimally aligned have 95o amino acid homology. As used herein, homology refers to identical amino acids or residue positions which are not identical but differ only by conservative amino acid substitutions. For example, 25 the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to affect the properties of a protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid.
30 The phrase "substantially purified" or "isolated"
when referring to a protein (or peptide), means a chemical composition which is essentially free of other cellular components. It is preferably in a homogeneous state although it can be in either a dry or aqueous WO 00/15845 PCTIUS99Ii9.b75 solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein (or peptide) which is 5 the predominant species present in a preparation is -substantially purified. Generally, a substantially purified or isolated protein (or peptide) will comprise more than 80% of all macromolecular species present in the preparation. Preferably; the protein (or peptide) is purified to represent greater than 90% of all macromolecular species present. l~iore preferably the protein (or peptide) is purified i.o greater than 950, and most preferably the protein (or peptide) is purified to essential homogeneity, wherein other macromolecular 15 species are not detected by COnVeTltional techniques. A
"substantially purified" or "isolated" protein (or peptide) can be separated from an organism, synthetically or chemically produced, or recombLnantly produced.
"Biological sample" or "samp7Le" as used herein 20 refers to any sample obtained from a living organism or from an organism that has died. Examples of biological samples include body fluids and tissue specimens.
High stringent hybridization conditions are selected at about 5°C lower than the thermal melting point (Tm) 25 for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of t:he target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those: in which the salt 30 concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may significantly affect the stringency of hybridization, including, among athers, base camp>osition and size of the complementary strands, the presence of organic solvents, WO 00115845 PCT/US99ft9675 i.e. salt or formamide concentrat_Lon, and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one. High stringency may be attained, for example, by overnight hybridization at about 68°C in a E~X SSC solution, washing-at room temperature with 6X SSC solution, followed by washing at about 68°C in a 6X SSC solution then in a 0.6X
SSX solution.
Hybridization with moderate :stringency may be attained, for example, by: 1) filter pre-hybridizing and hybridizing with a solution of 3X sodium chloride, sodium citrate (SSC) , 50% formamide, 0.112 Tris buffer at pH 7.5, 5X Denhardt's solution; 2) pre-hybridization at 37°C for 4 hours; 3) hybridization at 37°C with amount of labeled probe equal to 3,000,000 cpm total. for 16 hours; 4) wash in 2X SSC and O.lo SDS solution; 5) wash 4X for 1 minute each at room temperature and 4X at. 60°C for 30 minutes each; and 6) dry and expose to film.
The phrase "selectively hybridizing to" refers to a nucleic acid molecule that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or R.NA. By selectively hybridizing it is meant that a nucleic acid molecule: binds to a given target in a manner that is detectable in a different manner from non-target sequence under moderate, or more preferably under high, stringency conditions of hybridization. "Complementary" or' "target" nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid molecule. Proper annealing conditions depend, for example, upon a nucleic acid molecule's length, base composition, and the number of mismatches and their position on the molecule, and must often be determined empirically. For discussions of WO 00/15845 PCT/US99/t9675 nucleic acid molecule (probe) de~~ign and annealing conditions, see, for example, Sarribrook et al. 1989.
It will be readily understoc>d by those skilled in the art and it is intended here, that when reference is 5 made to particular sequence listings, such reference -includes sequences which substantially correspond to its complementary sequence and those described including allowances for minor sequencing errors, single base changes, deletions, substitutions and the like, such that 10 any such sequence variation corresponds to the nucleic acid sequence of the signal peptide or other peptide/protein to which the relevant sequence listing relates.
The DNA molecules of the subject invention also 15 include DNA molecules coding for protein analogs, fragments or derivatives of the protein which differ from naturally-occurring forms (the naturally-occurring protein) in terms of the identity or location of one or more amino acid residues (deletion analogs containing 20 less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other ressidues, and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the protein) and which 25 share the signal property of the naturally-occurring form. These molecules include: tree incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymE;s; and the provision of 30 additional initial, terminal or intermediate DNA
sequences that facilitate construction of readily expressed vectors.
As used herein, a "peptide" refers to an amino acid sequence of three to one hundred amino acids, and therefore an isolated peptide that comprises an amino acid sequence is not intended to cover amino acid sequences of greater than 100 amino acids. Preferably, the peptides that can be identified and used in 5 accordance with the subject invention {whether they be -mimotope or anti-mimotope peptides} are less than 50 amino acids in length, and more preferably the peptides are five to 20 amino acids in length or 20-40 amino acids in length.
10 The peptides can contain any naturally-occurring or non-naturally-occurring amino acids, including the D-form of the amino acids, amino acid derivatives and amino acid mimics, so long as the desired function and activity of the peptide is maintained. The choice of including an 15 (L)- or a (D)-amino acid in the peptides depends, in part, on the desired characteris tics of the peptide. For example, the incorporation of one or more (D)-amino acids can confer increased stability on the peptide and can allow a peptide to remain active :in the body for an 20 extended period of time. The incorporation of one or more (D)-amino acids can also increase or decrease the pharmacological activity of the peptide.
The peptides may also be cyclized, since cyclization may provide the peptides with superior properties over 25 their linear counterparts.
As used herein, the terms "arnino acid mimic" and "mimetic" mean an amino acid analog or non-amino acid moiety that has the same or similar functional characteristic of a given amino acid. For instance, an 30 amino acid mimic of a hydrophobic amino acid is one which is non-polar and retains hydrophok>icity, generally by way of containing an aliphatic chemical group. -By way of further example, an arginine mimic: can be an analog of arginine which contains a side chain having a positive WO 00/15845 PCTIUS99/i-9675 _ 21 _ charge at physiological pH, as is characteristic of the guanidinium side chain reactive group of arginine.
In addition, modifications to the peptide backbone and peptide bonds thereof are also encompassed within the scope of amino acid mimic or mimetic. Such modifications-can be made to the amino acid, derivative thereof, non-amino acid moiety or the peptide either before or after the amino acid, derivative thereof or non-amino acid moiety is incorporated into the peptide. What is critical is that such modifications mimic the peptide backbone and bonds which make up 'the same and have substantially the same spacial arrangement and distance as is typical for traditional pepi~ide bonds and backbones. An example of one such modification is the reduction of the carbonyls) of the amide peptide backbone to an amine. A number oi= reagents are available and well known for the reduction of amides to amines such as those disclosed in Wann et al., JOC, 46:257 (1981) and Rancher et al., Tetrahedron. Lett.,, 21:14061 (1980). An amino acid mimic is, therefor, an organic molecule that retains the similar amino acid pharmacophore groups as is present in the corresponding amino acid and which exhibits substantially the same spatial arrangement between functional groups.
The substitution of amino ac~.ds by non-naturally occurring amino acids and amino acid mimics as described above can enhance the overall activity or properties of an individual peptide based on thE: modifications to the backbone or side chain functionalities. For example, these types of alterations to the specifically described amino acid substituents and exemplified peptides can enhance the peptide's stability to enzymatic breakdown and increase biological activity. Modifications to the WO 00/15$45 PCTIUS99/t9675 peptide backbone similarly can add stability and enhance activity.
One skilled in the art, using the above sequences or formulae, can easily synthesize tlhe peptrides. Standard procedures for preparing synthetic peptides are well known in the art. The novel peptides can be synthesized using: the solid phase peptide synthesis (SPPS) method of Merrifield (J. Am. Chem. Soc., 85:2149 (1964)) or modifications of SPPS; or, the peptides can be synthesized using standard solution methods well known in the art (see, for example, Bodanz~;ky, M., Principles of Peptide Synthesis, 2nd revised ed., Springer-Verlag (1988 and 1993)). Alternatively, simultaneous multiple peptide synthesis (SMPS) techniques well l~,nown in the art can be used. Peptides prepared by the method of Merrifield can be synthesized using an. automated peptide synthesizer such as the Applied Biosystems 431.A-O1 Peptide Synthesizer (Mountain View, Calif.) or using the manual peptide synthesis technique described by Houghten, Proc.
Natl. Acad. Sci., USA 82:5131 (1985).
With these definitions in mind, the subject invention provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel. The nucleic acid molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA, including messenger RNA or mRNA), genomic or recombinant, biologically isolated or synthetic.
The DNA molecule can be a cDNA molecule, which is a DNA copy of a messenger RNA (mRNA) encoding the channel.
An example of such a pancreatic T-type calcium channel is the rat pancreatic T-type calcium channel encoded by the nucleotide sequence as shown in SEQ ID
NO:1. The amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID N0:2.

WO 00/15845 PCTlUS99/19675 _ 23 _ _ The invention also provides an antisense nucleic acid molecule that is complementary to at least a portion of the mRNA encoding the pancreatic T-type calcium channel. Antisense nucleic acid molecules can be RNA or 5 single-stranded DNA, and can be complementary to the -entire mRNA molecule encoding the channel (i.e. of the same nucleotide length as the entire molecule). It may be desirable, however, to work with a shorter molecule.
In this instance, the antisense molecule can be 10 complementary to a portion of the entire mRNA molecule encoding the channel. These shorter antisense molecules are capable of hybridizing to the mRNA encoding the entire molecule, and preferably consist of about twenty to about one hundred nucleotides. These antisense 15 molecules can be used to reduce lcavels of pancreatic T-type calcium channel, by introduc:i:ng into cells an RNA or single-stranded DNA molecule that is complementary to at least a portion of the mRNA of thE~ channel (i.e. by introducing an antisense molecule). The antisense 20 molecule can base-pair with the mFtNA of the channel, preventing translation of the mRNA into protein. Thus, an antisense molecule to the channel can prevent translation of mRNA encoding the channel into a functional channel protein. It may be desirable to place 25 the antisense molecule downstream and under the control or the insulin promoter, so that t:he antisense will prevent translation of mRNA encoding the T type calcium channel only in islet cells of they pancreas (not affecting brain or heart T type calcium channels). It 30 should also be apparent that 100% prevention of T type calcium channel is not desirable, since a minimal basal Caz+ level is required to be maintained by the T type calcium channel.

WO OOI15845 PCT/US99It9b75 _ 24 More particularly, an antiserzse molecule complementary to at least a portic>n of mRNA encoding a pancreatic T-type calcium channel can be used to decrease expression of a functional channel.. A cell with a first level of expression of a functional pancreatic T-type -calcium channel is selected, and then the antisense molecule is introduced into the cell. The antisense molecule blocks expression of functional pancreatic T-type calcium channel, resulting in. a second level of expression of a functional pancreatic T-type calcium channel in the cell. The second level is less than the initial first level.
Antisense molecules can be introduced into cells by any suitable means. In one embodiment, the antisense RNA
15, molecule is injected directly into the cellular cytoplasm, where the RNA interferes with translation. A
vector may also be used for introduction of the antisense molecule into a cell. Such vectors include various plasmid~and viral vectors. For a general discussion of antisense molecules and their use, see Han et al. 1991 and Rossi 1995.
The invention further provides a special category of antisense RNA molecules, known as ribozymes, having recognition sequences complementary to specific regions of the mRNA encoding the pancreatic T-type calcium channel. Ribozymes not only complex with target sequences via complementary antise:nse sequences but also catalyze the hydrolysis, or cleavage, of the template mRNA molecule. Examples, which are not intended to be limiting, of suitable regions of t:he mRNA template to be targeted by ribozymes are any of t'.he homologous regions identified by comparing the various T-type calcium channels, and particularly pancreatic ~i-cell T-type channels.

WO 00/15845 PCT/US99f19l75 Expression of a ribozyme in <~ cell can inhibit gene expression (such as the expression of a pancreatic T-type calcium channely. More particularly, a ribozyme having a recognition sequence complementar~,r to a region of a mRNA
encoding a pancreatic T-type calc:Lum channel can be used.-_ to decrease expression of pancreatic T-type calcium channel. A cell with a first level of expression of pancreatic T-type calcium channel is selected, and then the ribozyme is introduced into the cell. The ribozyme in the cell decreases expression of pancreatic T-type calcium channel in the cell, because mRNA encoding the pancreatic T-type calcium channel is cleaved and cannot be translated.
Ribozymes can be introduced 9.nto cells by any suitable means. In one embodiment:, the ribozyme is injected directly into the cellular cytoplasm, where the ribozyme cleaves the mRNA and thereby interferes with translation. A vector may be used for introduction of the ribozyme into a cell. Such vectors include various plasmid and viral vectors (note that the DNA encoding the ribozyme does not need to be "incorporated" into the genome of the host cell; it could be expressed in a host cell infected by a viral vector, with the vector expressing the ribozyme, for instance). For a general discussion of ribozymes and their use, see Sarver et al.
1990, Chrisey et al. 1991, Rossi e:t al. 1992, and Christoffersen et al. 1995.
The nucleic acid molecules of the subject invention can be expressed in suitable host cells using conventional techniques. Any suitable host and/or vector system can be used to express the pancreatic T-type calcium channel. For in vitro expression, Xenopus oocytes are preferred. For in vivo expression, the most suitable host cell is a pancreatic (3-cell.

WO 00/15845 PCTIUS99/~19575 Techniques forintroducing the nucleic acid molecules into the host cells may involve the use of expression vectors which comprise the nucleic acid molecules. These expression vectors (such as plasmids 5 and viruses; viruses including bacteriophage) can then be-used to introduce the nucleic acid molecules into suitable host cells. For example, DNA encoding the pancreatic T-type calcium channel can be injected into the nucleus of a host cell or transformed into the host cell using a suitable vector, or mRNA encoding the pancreatic T-type calcium channel can be injected directly into the host cell, in order to obtain expression of pancreatic T-type calcium channel in the host cell.
15 Various methods are known in the art for introducing nucleic acid molecules into host cells. One method is microinjection, in which DNA is injected directly into the,nucleus of cells through tine glass needles (or RNA
is injected directly into the cytoplasm of cells).
20 Alternatively, DNA can be incubated with an inert carbohydrate polymer (dextran) to which a positively charged chemical group (DEAE, for diethylaminoethyl) has been coupled. The DNA sticks to i~he DEAE-dextran via its negatively charged phosphate groin?s. These large DNA-25 containing particles stick in turn to the surfaces of ce2ls, which are thought to take them in by a process known as endocytosis. Some of they DNA evades destruction in the cytoplasm of the cell and escapes to the nucleus, where it can be transcribed into F2NA like any other gene 30 in the cell. In another method, cells efficiently take in DNA in the form of a precipitate with calcium phosphate. In electroporation, cells are placed in a solution containing DNA and subjecaed to a brief electrical pulse that causes hole:> to open transiently in their membranes. DNA enters through the holes directly into the cytoplasm, bypassing the endocytotic vesicles through which they pass in the DEAE-dextran and calcium phosphate procedures. DNA can ale>o be incorporated into artificial lipid vesicles, liposomes, which fuse with the -cell membrane, delivering their cc>ntents directly into the cytoplasm. In an even more direct approach, DNA is absorbed to the surface of tungsten microprojectiles and fired into cells with a device re~:embling a shotgun.
Several of these methods, microinjection, electroporation, and liposome fusion, have been adapted to introduce proteins into cells. For review, see Mannino and Gould-Fogerite 1988, Shigekawa and Dower 1988, Capecchi 1980, and Klein et al. 1987.
Further methods for introducing nucleic acid molecules into cells involve the use of viral vectors.
One such virus widely used for protein production is an insect virus, baculovirus. For a review of baculovirus vectors, see Miller (1989). Various viral vectors have also been used to transform mammalian cells, such as bacteriophage, vaccinia virus, adenovirus, and retrovirus.
As indicated, some of these methods of transforming a cell require the use of an intermediate plasmid vector.
U.S. Patent No. 4,237,224 to Cohen and Boyer describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
The DNA sequences are cloned into 'the plasmid vector using standard cloning procedures :known in the art, as described by Sambrook et al. (1989).

WO 00!15845 PCT/US99fi9675 - 2$
Host cells into which the nucleic acid encoding the pancreatic T-type calcium channel has been introduced can be used to produce (i.e. to functionally express) the pancreatic T-type calcium channel. The function of the 5 encoded pancreatic T-type calcium channel can be assayed -according to methods known in the art (Wang et al. 1996).
Having identified the nucleic acid molecules encoding pancreatic T-type calcium channels and methods for expressing the pancreatic T-type calcium channels 10 encoded thereby, the invention further provides a method of screening a substance (for example, a compound or inhibitor) for the ability of the substance to modify T-type calcium channel function. The method comprises introducing a~nucleic acid molecule encoding the 15 pancreatic T-type calcium channel into a host cell, and expressing the pancreatic T-type calcium channel encoded by the molecule in the host cell. The cell is then exposed to a substance and evaluated to determine if the substance modifies the function of: the T-type calcium 20 channel. From this evaluation, substances effective in altering the function of the T-type calcium channel can be found. Such agents tray be, for example, calcium channel inhibitors, agonists, or antagonists (for example, mibefradil and mibefradil. analogues, amiloride, 25 NiCl2, antisense molecules, and second messengers).
The evaluation of the cell to determine if the substance modifies the function of the T-type calcium channel can be by any means known in the art. The evaluation can comprise the direct. monitoring of 30 expression of T-type calcium channel in the host cell, or the evaluation can be indirect anf. comprise the monitoring of calcium transport by the channel (such as by the methods disclosed by Wang et al. 1996).

WO 00/15845 PCT/US991i9b75 The nucleic acid molecules of the subject invention can be used either as probes or for the design of primers to obtain DNA encoding other pancreatic T-type calcium channels by either cloning and colony/plaque 5 hybridization or amplification using the polymerase chair3-reaction (PCR).
Specific probes derived from SEQ TD NO:1 can be employed to identify colonies or :plaques containing cloned DNA encoding a member of t:he pancreatic T-type 10 calcium channel family using known methods (see Sambrook et al. 1989}. One skilled in the art will recognize that by employing such probes under high stringency conditions (for example, hybridization at 42°C with 5X SSPC and 50%
formamide, washing at 50-65°C with O.SX SSPC}, sequences 15 having regions which are greater 'than 90% homologous or identical to the probe can be obtained. Sequences with lower percent homology or identit~~ to the probe, which also encode pancreatic T-type calcium channels, can be obtained by lowering the stringency of hybridization and 20 washing (e. g., by reducing the hybridization and wash temperatures or reducing the amount of formamide employed).
More particularly, in one embodiment, the method comprises selection of a DNA molecule encoding a 25 pancreatic T-type calcium channel" or a fragment thereof, the DNA molecule having a nucleotide sequence as shown in SEQ TD N0:1, and designing an oli.gonucleotide probe for pancreatic T-type calcium channel based on SEQ ID NO:1.
A genomic or cDNA library of an organism is then probed 30 with the oligonucleotide probe, and clones are obtained from the library that are recognized by the oligonucleotide probe so as to obtain DNA encoding another pancreatic T-type calcium channel.

WO 00115845 PCT/US99H9b75 Specific primers derived from SEQ ID NO:1 can be used in PCR to amplify a DNA sequence encoding a member of the pancreatic T-type calcium channel family using known methods (see Innis et al. 1990). One skilled in 5 the art will recognize that by employing such primers -under high stringency conditions (for example, annealing at 50-50°C, depending on the lengi~h and specific nucleotide content of the primers employed), sequences having regions greater than 75% homologous or identical to the primers will be amplified.
More particularly, in a further embodiment the method comprises selection of a DIVA molecule encoding pancreatic T-type calcium channel, or a fragment thereof, the DNA molecule having a nucleotide sequence as shown in 15 SEQ ID N0:1, designing degenerate oligonucleotide primers based on regions of SEQ ID NO:1, and employing such primers in the polymerase chain reaction using as a template a DNA sample to be screened for the presence of pancreatic T-type calcium channe l-encoding sequences.
20 The resulting PCR products can be isolated and sequenced to identify DNA fragments that encode polypeptide sequences corresponding to the targeted region of pancreatic T-type calcium channel.
Various modifications of the nucleic acid and amino 25 acid sequences disclosed herein are covered by the subject invention. These varied sequences still encode a functional pancreatic T-type calcium channel. The invention thus further provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel, 30 the nucleic acid molecule encoding a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence, the sE.cond amino acid sequence as shown in SEQ ID N0:2. In further embodiments, the first amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% amino acid identity to SEQ ID
N0:2.
The invention further provides an isolated DNA
oligomer capable of hybridizing t.o the nucleic acid molecule encoding pancreatic T-type calcium channel according to the subject invention. Such oligomers can be used as probes in a method of detecting the presence of pancreatic T-type calcium channel in a sample. More particularly, a sample can be contacted with the DNA
10 oligomer and the DNA oligomer will. hybridize to any pancreatic T-type calcium channel present in the sample, forming a complex therewith. The complex can then be detected, thereby detecting presence of pancreatic T-type calcium channel in the sample.
The complex can be detected LESing methods known in the art. Preferably, the DNA olic~omer is labeled with a detectable marker so that detection of the marker after the DNA oligomer hybridizes to any pancreatic T-type calcium channel in the sample (wherein non-hybridized DNA
20 oligomer has been washed away) is detection of the complex. Detection of the complex: indicates the presence of pancreatic T-type calcium channel in the sample. As will be readily apparent to those skilled in the art, such a method could also be used quantitatively to assess the amount of pancreatic T-type calcium channel in a sample.
For detection, the oligomers can be labeled with;
for example, a radioactive isotope, biotin, an element opaque to X-rays, or a paramagnetic ion. Radioactive isotopes are commonly used and area well known to those skilled in the art. Representative examples include indium-111, technetium-99m, and iodine-123. Biotin is a standard label which would allow detection of the biotin labeled oligomer with avidin. Paramagnetic ions are also WO 00/15845 PCT/US99Ji-9675 - 32 - _ _ commonly used and include, for example, chelated metal ions of chromium (III), manganese (II), and iron (III).
When using such labels, the labeled DNA oligomer can be imaged using methods known to those skilled in the art.
Such imaging methods include, but are not limited to, X- -ray, CAT scan, PET scan, NMRI, and. fluoroscopy. Other suitable labels include enzymatic labels (horseradish peroxidase, alkaline phosphatase, etc.) and fluorescent labels (such as FITC or rhodamine, etc.).
The invention further provides an isolated pancreatic T-type calcium channel protein. The protein is preferably encoded by a nucleotide sequence as shown in SEQ ID NO: 1. The protein preferably has an amino acid sequence as shown in SEQ ID N0:2. Further provided is an isolated pancreatic T-type calcium channel protein encoded by a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence, the second amino acid sequence as shown in SEQ ID N0:2.
In further embodiments, the first .amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% amino acid identity to SEQ ID N0:2.
The pancreatic T-type calcium channel molecule of the subject invention can include a leader sequence for targeting of the pancreatic T-type calcium channel protein to the desired part of a cell.
It should be readily apparent to those skilled in the art that a met residue may need to be added to the amino terminal of the amino acid sequence of the mature pancreatic T-type calcium channel protein (i.e., added to SEQ ID N0:2) or an ATG added to the=_ 5' end of the nucleotide sequence (i.e., added to SEQ ID NO:1), in order to express the channel in a host cell. The met version of the mature channel is thus specifically WO UO/IS$45 PCTlUS99/t9675 intended to be covered by reference to SEQ ID N0:1 or SEQ
ID N0:2.
The invention further provides an antibody or fragment thereof specific for the pancreatic T-type calcium channel of the subject invention. Antibodies of -the subject invention include pol~rclonal antibodies and monoclonal antibodies capable of binding to the pancreatic T-type calcium channel,, as well as fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the sux>ject invention may be generated using one of the procedures known in the art such as chimerization. Fragments of the antibodies of the present invention include, but: are not limited to, the Fab, the F(ab')2; and the Fc fragments.
The invention also provides hybridomas which are capable of producing the above-de~;cribed antibodies. A
hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (see Campbell 1984 and St. troth et al. 1980). Any animal (mouse, rabbit, etc.) which. is known to produce antibodies can be immunized with the antigenic pancreatic T-type calcium channel (or an antigenic fragment thereof). Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the protein. One skilled in the art will recognize that the amount of the protein used for immunization will vary based on the animal which is immunized, the antigenicity of the protein, and the site of injection.
The protein which is used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicit.y. Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as a globulin ar beta-galactosidase) ox' through the inclusion -of an adjuvant during immunization..
For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag 15 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
Any one of a number of methods well known in the art can be used to identify the hybrid.oma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al.
1988) .
F~iybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell 1984).
For polyclonal antibodies, antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
The present invention further provides the above-described antibodies in detectably labeled form:
Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc.
Procedures for accomplishing such labeling are well known in the art, for example see Sternberger et al. 1970;
Bayer et al. 1979, Engval et al. 1972, and Goding 1976.

WO 00115$45 PCT/U5991~91>75 The labeled antibodies or fragments thereof of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues which express pancreatic T-type calcium channel,; to identify samples containing pancreatic T-type calcium channel, or to -detect the presence of pancreatic T-type calcium channel in a sample. More particularly, t:he antibadies or fragments thereof can thus be used to detect the presence of pancreatic T-type calcium chanrzel in a sample, by contacting the sample with the antibody or fragment thereof. The antibody or fragment thereof binds to any pancreatic T-type calcium channel present in the sample, forming a complex therewith. The complex can then be detected, thereby detecting the presence of pancreatic T-type calcium channel in~the sample. As will be readily apparent to those skilled in the art, such a method could also be used quantitatively to as~~ess the amount of pancreatic T-type calcium channel in a sample. As should also be readily apparent, such an antibody can also be used to decrease levels of functic>nal T type calcium channels, by blocking the channel. Such antibodies can therefore be used in the methods of the subject invention to modify levels of functional T type calcium channels in pancreatic beta cells.
Further provided is a composition comprising the pancreatic T-type calcium channel protein and a compatible carrier.
In the methods of the invention, tissues or cells are contacted with or exposed to the composition of the subject invention or a compound. In the context of this invention, to "contact" tissues or cells with or to "expose" tissues or cells to a composition or compound means to add the composition or compound, usually in a liquid carrier, to a cell suspension or tissue sample, WO 00/15845 PCT/US99/~9675 - 36 _ either in vitro or ex vivo, or to administer the composition or compound to cells or tissues within an animal (including humans).
For therapeutics, methods of modifying insulin secretion by pancreatic beta cell~~, methods of treating -type TI diabetes, methods of modifying basal calcium levels in cells, methods of modifying the action potential of L type calcium channE~ls in cells, methods of modifying pancreatic beta cell death, methods of modifying pancreatic beta cell proliferation, and methods of modifying calcium influx through L type calcium channels in cells, each of the methods comprising modifying levels of functional T type calcium channels in the cells, are provided. The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill in the art. In general, for therapeutics, a patient suspected of needing such therapy is given a composition in accordance with the invention, commonly in a pharmaceu.t~.cally acceptable carrier, in amounts and far periods which will vary depending upon the nature of the particular disease, its severity and the patient's overall condition. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intrathecal or intraventricular administration.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like maybe necessary or desirable. Coated condoms, gloves and the like may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-10 aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Compositions for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
In addition to such pharmaceutical carriers, cationic lipids may be included in the formulation to facilitate uptake. One such composition shown to facilitate uptake is LIPOFECTTN (BRL; Bethesda MD).
Dosing is dependent on severity and responsiveness of the condition.to be treated, with course of treatment lasting from several days to Several months or until a cure is effected or a diminution of disease state is 25 achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body.
Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
Optimum dosages may vary depending on the relative 30 potency of individual composition~~, and can generally be calculated based on ICso' s or ECso' ;s in in vitro and in vivo animal studies. For example, given the molecular weight of compound (derived from oligonucleotide sequence and/or chemical structure ) and an effective dose such as WO 00115845 PCTIUS99/~ 9_675 an ICso, for example (derived experimentally), a dose in mg/kg is routinely calculated.
The methods of the subject invention are based on the discovery that regulation of 'T type calcium channels 5 directly modifies basal calcium levels in cells, which in turn regulates L type calcium channel activity, which in turn regulates insulin secretion <~nd cell death, which in turn treats type II diabetes. Thc~ methods of the subject invention are further based on thE= discovery that 10 regulation of T type calcium channels directly affects basal and glucose-induced insulin secretion.
T type calcium channels belong to the family of low voltage activated calcium channel:. Modifying (increasing or decreasing) "levels" of functional T type 15 calcium channels refers to modify_Lng expression of the T
type calcium channel gene, modifying activity of the T
type calcium channel such as by inhibiting the function of the channel, and/or modifying t:he formation of active membrane-spanning T type calcium channels. As used 20 herein, "functional" refers to the synthesis and any necessary post-translational processing of a calcium channel molecule in a cell so that: the channel is inserted properly in the cell membrane and is capable of conducting calcium ions in accordance with a low voltage 25 activated channel.
The invention thus provides a method of modifying insulin secretion by pancreatic beta cells, the method comprising modifying levels of T type calcium channels in the pancreatic beta cells.
30 Levels of T type calcium channels in the pancreatic beta cells can be modified by various methods, at the gene and protein and °functional calcium channel" levels.
In one embodiment, the levels are modified by modifying T
type calcium channel gene expression of the T type WO 00115845 PCT/US99t19675 calcium channel in the cells. This can be accomplished by exposing the cells to a compound which modifies T type calcium channel gene expression of the calcium channel.
The compound could be, for example, an antisense 5 oligonucleotide targeted to the T type calcium channel -gene. In a similar embodiment, t:he compound which modifies T type calcium channel gene expression of the T
type calcium channel could be a ribozyme.
Other methods for modifying 'T type calcium channel gene expression could also involve site-directed mutagenesis of the T type calcium channel gene to prevent expression of the T type calcium channel, or various gene therapy techniques.
Levers, in particular activity, of T type calcium 15 charmers in the cell can also be modified by exposing the cells to an inhibitor of the T type calcium channel.
Such inhibitors include, for exams?1e, mibefradil, mibefradil analogs, amiloride, Nit~lz, and second messengers which regulate activity of the T type calcium 20 channels. Other inhibitors of they T type calcium channel could arso readily be identified by screening methods (including the method described above). In addition to chemicar inhibitors, peptide inhibitors could also be identified with screening methods (for example, using 25 phage display libraries and other peptide screening methods ) .
"Mibefradil analogs", as used herein are meant to include compounds having the formula:

WO 00/15845 PCT/US991-t9675 H3C~, CH 3 / R
O_ N

.s wherein R is hydrogen, alkyl, or a moiety having the formula C(O)R', where R' is alkyl or aryl. In the above formulae, alkyl is meant to include linear alkyls, particularly C1-CI2 linear alkyls (e. g., methyl, ethyl, n-propyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and the like), branched alkyls, particularly C1-C12 branched alkyls (e. g., isobutyl, isopentyl, neopentyl, hex-2-yl, hex-3-yl, hept-2-yl, hep~t-3-yl, and the like), and cycloalkyls, particularly C1-C'8 cycloalkyls (e. g., cyclopentyl, cyclohexyl, cycloheptyl, 4-methylcyclohexyl, and the like). These alkyl groups can be substituted or unsubstituted. When substituted, suitable substituents include, for example, aryl groups, halogen atoms, hydroxy groups, alkoxy groups, carboxylic acid groups, amine groups, and the like, as well as combinations of these substituents. Mibefradil analogs which are particularly well suited to blocking (inhibiting) the activity of T-type calcium channels but not blocking the activity of L-type calcium channels are those having the formula:
H3C~CH3 OH
N
i ~''~ \
F ~ CH3 N

WO 00/15845 PCT/U599t1~675 and those having the formula:

H3C~CH3 R,.
O -N
F ~ CH3 N
~r~--5 in which R" is an unsubstituted alkyl group or a substituted alkyl group which does not contain an alkoxy substituent. "Mibefradil analogs" are also meant to include compounds having the above formulae which are substituted at other positions in the structure, for 10 example, on the benzimidazole phenyl moiety, at a benzimidazole nitrogen, at other positions of the tetrahydronaphthyl ring, etc. Also included within the meaning of "mibefradil analogs" are compounds having the above formulae in which the F is replaced with another 15 substituent, such as another halogen. Also included within the meaning-of "mibefradil analogs" are compounds having the above formulae in which the amine methyl group or the isopropyl group or both are' replaced with other substi,tuents, such as other alkyl moieties.
20 Additionally, "mibefradil analogs" are meant to include those compounds which are generically described and/or specifically disclosed in U.S. Patient No. 4,808,605, which is hereby incorporated by reference. Further, "mibefradil analogs" are meant to include 25 pharmaceutically acceptable salts of the derivatives described above. Illustrative pharmaceutically acceptable salts are salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid, phosphoric acid, citric acid, formic acid, malefic acid, acetic acid, succinic acid, tartaric acid, methanesulphonic acid, p-toluenesulphonic, and the like.
Mibefradil analogs can be made by following the general procedures described in, :Eor example, U.S. Patent Nos. 4,808,605, 5,910,606, 5,892,1)55, 5,811,557, 5,811,556, and 5,808,088, each of which is hereby incorporated by reference.
Levels of T type calcium channels in the cell can also be modified by exposing the cells to a compound which interferes with membrane T type calcium channel formation.
Levels of functional T type calcium channel could also be modified by use of molecu7.es which bind to transcription regulators of the T type calcium channel gene (such as the promoter region of the gene).
The invention further provides a method of treating type TI diabetes in a subject (human or animal), the method comprising administering to the subject an amount of a compound effective to modify levels of T type calcium channels in the pancreatic' beta cells of the subject. As above, the compound may modify levels of T
type calcium channels by modifying T type calcium channel gene expression of the calcium channel, or by inhibiting the T type calcium channel, or by interfering with membrane T type calcium channel formation.
In the context of this invention "modulation" or "modifying" means either inhibition or stimulation. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA
expression, Western blot assay of protein expression, or calcium channel activity assay.
The compounds and/or inhibitors used in the methods of the subject invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound/inhibitor which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, S the disclosure is also drawn to p:rodrugs and -pharmaceutically acceptable salts of the compounds and/or inhibitors used in the subject invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
In regard to prodrugs, the compounds and/or izihibitors for use in the invention may additionally or alternatively be prepared to be delivered in a prodrug form. The term prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
In regard to pharmaceutically acceptable salts, the term pharmaceutically acceptable :alts refers to physiologically and pharmaceutica7_ly acceptable salts of the compounds and/or inhibitors u~~ed in the subject invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
Drugs, such as peptide drugs, which inhibit the T
type calcium channel or which interfere with functional T
type calcium channel formation can be identified by other methods also. For example, a monc>clonal antibody can be prepared which specifically hybridizes to the T type calcium channel, thereby interfering with activity and/or channel formation. Once a monoclonal antibody which specifically hydridizes to the T type calcium channel is identified, the monoclonal (which is itself a compound or inhibitor which can be used in the subject invention) can be used to identify peptides capable of mimicking the inhibitory activity of the monoclonal antibody. One such method utilizes the development of epitope libraries and biopanning of bacteriophage libraries. Briefly, attempts 5 to define the binding sites for various monoclonal -antibodies have led to the development of epitope libraries. Parmley and Smith developed a bacteriophage expression vector that could display foreign epitopes on its surface (Parmley, S.F. & Smith, G.P., Gene 73:305-318 10 (1988)). This vector could be used to construct large collections of bacteriophage which could include virtually all possible sequences of a short (e. g: six-amino-acid) peptide. They also developed biopanning, which is a method for affinity-purifying phage displaying 15 foreign epitapes using a specific antibody (see Parmley, S.F. & Smith, G.P., Gene 73:305-3:18 (1988); Cwirla, S.E., et al., Proc Natl Acad Sci USA 87:6378-6382 (1990);
Scott, J.K. & Smith, G.P., Science 249:386-390 (1990);
Christian, R.B., et al., J Mol Biol 227:711-718 (1992);
20 Smith, G.P. & Scott, J.K., Methods in Enzymology 217:228-257 (1993)}.
After the development of epii~ope libraries, Smith et al. then suggested that it should be possible to use the bacteriophage expression vector and biopanning technique 25 of Parmley and Smith to identify E~pitopes from all possible sequences of a given len<~th. This led to the idea of identifying peptide ligands for antibodies by biopanning epitope libraries, which could then be used in vaccine design, epitope mapping, t:he identification of 30 genes, and many other applications (Parmley, S.F. &
Smith, G.P., Gene 73:305-318 (198E3); Scott, J.K., Trends in Biochem Sci 17:241-245 (1992))., Using epi.tope libraries and biopanning, researchers searching for epitope sequences found instead peptide sequences which mimicked the epitppe, i.e., sequences which did not identity a continuous linear native sequence or necessarily occur at all withixi a natural protein sequence. These mimicking peptides are called 5 mimotopes. In this manner, mimotapes of various binding._ sites/proteins have been found.
The sequences of these mimotopes, by definition, do not identify a continuous linear native sequence or necessarily occur in any way in a naturally-occurring 10 molecule, i.e. a naturally occurring protein. The sequences of the mimotopes merely form a peptide which functionally mimics a binding site on a naturally-occurring protein.
Many of these mimotapes are :short peptides. The 15 availability of short peptides which can be readily synthesized in large amounts and vahich can mimic naturally-occurring sequences (i.e. binding sites) offers great potential application.
Using this technique, mimotopes to a monoclonal 20 antibody that recognizes T type calcium channels can be identified. The sequences of these mimotopes represent short peptides which can then be used in various ways, for example as peptide drugs that bind to T type calcium channels and decrease the activity of T type calcium 25 channels. Once the sequence of the mimotope is determined, the peptide drugs can be chemically synthesized.
MATERTALS AND METHODS
30 Cell Culture - INS-1 cells were cultured in RPMI
1640 medium containing loo FBS, 25 U/ml penicillin, 25 mg/ml streptomycin and 50 ~,M mercaptoethanol in an atmosphere of 5% COz in air, at 37°C for 2-5 days before recording.

WO 00/15845 PCT/US99/19b75 Islet cell preparation - Pancreases of Sprague-Dawley rats (Charles River Laboratory, Wilmington, MA) were removed after intrapancreatic perfusion with 2 ml of Hanks' solution (Gibcci BRL, Grand 5 Island, NY) containing collagenase (4 mg/ml, Boehringer -Mannheim, Indianapolis, IN) , DNasE~ I (10 ~Cg/ml, Sigma, St. Louis, MO), CaCl2 (1.28 mM) anal bovine serum albumin (1 mg/ml, Gibco BRL). The pancreatic tissue was incubated at 37°C for 20 min and then washed five times with 10 enzyme-free Hanks' solution. Islets were picked up arid treated with 0.1% pancreatin (Sigma) for five minutes at 37°C. Single cells were obtained by triturating the islets with plastic pipette tips and then they were transferred into 35 mm culture di:~hes. Cells were 15 cultured in RPMI 1640 medium (Gibc:o BRL) containing 5 mM
glucose, 10% FBS and P/S at 37°C, 5% C02 for 2-5 days.
before experiments.
Isolation of RNA - Total RNA was isolated from cultured INS-1 cells and from various freshly excised rat 20 tissues by the guanidinium isothiocyanate/phenol procedure (Chomczynsk and Sacchi 1.987). Poly-A RNA was isolated from total RNA by two successive passes over an oligo (dT)-cellulose spin column (Arnbion, Austin, TX).
Cloning of cDNA Encoding al ~~ubunit of T-type Ca2+
25 channel in INS-1 - First strand cL>NA was prepared using 2 ~g of INS-1 cell mRNA and M-MLV reverse transcriptase (Gibco BRL) with the poly-dT prime:rs. The first 433 by DNA fragment of the channel was deduced with PCR using the degenerate primers (forward)(e~EQ ID N0:6) 5'-30 TNGC(A/C/T)ATGGAG(C/A)GNCC(C/T)~3' and (backward)(SEQ ID
NO: 7) 5' -CTT (C/G/T) CCCTTGAA(G/C)A(G/A) CTG) -3' based on conserved voltage-dependent Ca2+ channel al subunit sequences in domain III. Using the: MarathonT"' cDNA
Amplification Kit (Clontech, Palo Alto, CA), the 3!- and WO 00/15845 PCT/US99/19b75 5'- rapid amplifications of cDNA end-PCR (RACE-PCR) were performed to obtain the entire gene of the al subunit of the channel. For the 5'-RACE-PCR, the forward primer was an adapter primer, the backward primer was (SEQ ID N0:8}
5 5'-CCGCTGTCGGAGACCATGGAGACC-3'; for the 3'-RACE, the forward primer was (SEQ ID N0:9) 5'-AGCGGCCCAAAATTGACCCCCACAG-3' and 'the backward primer was poly-dT. The RT-PCR products were subcloned into pT-Adv Vector (Clontech) and dideoxynucl~~otide sequencing assay 10 was performed with a dsDNA Cycle ~5equencing System (Gibco BRL ) .
Tissue distribution -- The gene expression of T-type Ca2+ channels deduced from (3-cells was examined in rat brain, heart, kidney, and liver using an RT-PCR assay.
15 The primers used for the RT-PCR were {SEQ ID NO:10) 5'-GAAGATGCGAGTGGACAG-3' (forward,) and (SEQ ID NO:11) 5'-CTGTGGCGATGGTCACTG-3' (backward}. The PCR products were detected by agarose gel electrophoresis on a 1% gel.
Genome walking - The genome walker library 20 (Clontech) was used as a template in nested PCR reactions with gene-specific primers (GSP) and the adapter primers (AP) provided with the kit. The first PCR reaction was carried out in 5 tubes, each having a total volume of 50.1: 5~.1 10X PCR reaction buffer,, 1 ~,1 dNTP (10 mM
25 each) , 2 . 2~1 Mg (OAc) 2 (25 mM} . 1 N:1 AP1 (10 ~.M} , 1 ~.1 GSPl, 1 ~l Advantage Genomic Polyrnerase Mix (50X), and 37.8 ~.1 water. The following two-:step cycle parameters were used: (Step 1) 7 cycles of de=naturing at 94°C for 25 sec., annealing and extension at '72°C for 4 min. (Step 2) 30 32 cycles of denaturing at 94°C for 25 sec., annealing and extension at 67°C for 4 min. After the second step cycle, the samples were held at 6'7°C for 4 min. The second PCR reaction was carried out under the reaction condition similar to the first PCR reaction except using WO 00/15845 PCT/US991~9675 AP2, GSP2. In addition, the templates used were 1 ~l of 1:50 dilution of each primary PCR reaction. The two step cycles were similar to the first F?CR reaction except 5 cycles at the first step and 22 cycles at the second step. -Oocyte electrophysiology - cRNA transcripts were synthesized from BssH II linearized pT-Adv cDNA templates using T7 RNA polymerase (Ambion). Defolliculated Xenopus laevis were injected with 25 ng pT-Adv cRNA. Three to Z0 five days after injection, two-elE:ctrode voltage-clamp recording was performed using a Warner OC-725C amplifier (Warner Instrument Corp., Hamden, CT). Data were acquired and analyzed with Pulse/PulseFit :>oftware (HEKA, Lambrecht/Pfalz, Germany). The bath solution contained the following: 40 mM Ca (OH) z, 50 cnM NaOH, 2 mM TEA-C1, 1mM
KOH, 0.1 mM EDTA and S mM HEPES, adjusted to pH 7.4 with methanesulphonate. Boltzmann fits were calculated using Prism (GraphPad). Results are pre~~ented as mean ~ s.d.
unless otherwise stated.
(3-cell Electrophysiological recording - The whole-cell recordings were carried out by the standard "giga-seal" patch clamp technique (Hamill et al.). The whole-cell recording pipettes were made of hemocapillaries (Warner), pulled by a two-stage puller (PC-10, Narishige International, nfew York, NY), and heat polished with a microforge (MF200-1, World Precision Instruments, Sarasota, FL) before use. Pipette resistance was in the range of 2-5 MS2 in the internal solution. The recordings were performed at room temperature (22-25°C).
Currents were recorded using an EF~C-9 patch-clamp amplifier (HEKA) and filtered at 2.9 kHz. Data were acquired with Pulse/PulseFit software (HEKA).
Voltage-dependent currents were corrected for linear leak WO 00!15845 PCT/US99/39~75 and residual capacitance by using an on-line P/n subtraction paradigm.
Drugs - Mibefradil ( (1S, 2S} -2- [2- [ [3- (2-Benzimidazolyl)propyl]methyl-amino)ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-napthyl methoxy-acetate -dihydrochloride) was kindly provided by Dr. J.-P. Clozel (Hoffmann LaRoche, Basel, Switzerland),~and can be synthesized according to the methods disclosed in U.S.
Patent Nos. 5,892,055, 5,811,557, 5,811,556, and 10 5,808,088. U.S. Patent No. 4,808,,605 describes mibefradil compounds suitable for use in the subject invention.
The free alcohol Des-methoxyacetyl mibefradil (1S, 2S) -2- [2- [ [3- (2-Benzimidazoly7.) propyl]
15 methylamino]ethyl]-6-fluoro-1,2,3;4-tetrahydro-1-isopropyl-2-napthyl hydroxy hydrochloride) was prepared by alkaline hydrolysis: 14.2 mg mibefradil hydrochloride was dissolved in 4 ml methanol + 7. ml 10 N aqueous sodium hydroxide mixture (5 mM was the final concentration of 20 mibefradil). The solution was warmed in a boiling water bath for 10 min. The reaction was followed by mass spectrometry. Upon completion of t:he hydrolysis, as determined from the mass spectra, the solution was neutralized with 5 M aqueous hydrochloric acid. The 25 slight loss of methanol that occurred by evaporation during the reaction was corrected by adding water to keep the total volume of 5 ml.
Solutions - The extracellular solution used in whole-cell Ca2* current recording c:ontained.(in mM): 10 30 CaCl2, 110 tetraethylammonium-C1 ('TEA-C1), 10 CsCl, 10 N-2-hydroxyethylpiperazine-N'-2-etha.nesulfonic acid (HEPES), 40 sucrose, 0.5 3,4-diami.nopyridine, pH 7.3. The intracellular solution contained (in mM): 130 N-methyl-D-glucamine, 20 EGTA (free acid), 5 bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetate (BAPTA), 10 HEPES, 6 MgCl2, 4 Ca (OH) 2, pH was adjusted t:o 7 . 4 with methanesulfonate. 2 mM Mg-ATP was included in the pipette solution to minimize rundown of L-type Caz' currents. For Perforated-patch recording, the exa racellular solution -contained (in mM): 26 Sucrose, 30 TEA-Cl, 10 HEPES, 5 KC1, 2 CaCl2, MgCl2, pH 7.3. The pipette solution contained (in mM): 65 CsOH, 65 CsM:S, 20 sucrose, 10 HEPES, ZO MgCl2, 1 Ca (OH) 2, pH 7 . 4 .
Mass Spectrometric Analysis - A VG 70-250 SEQ
instrument (VG Analytical, Manchaster, UK) was used with fast atom bombardment (FAB) ionization mode to obtain mass spectra of the mibefradil and dm-mibefradil.
Cultured INS-1 cells were treated with 20 ~.M mibefradil for various lengths of time under each experimental condition. The cell pellets were collected after washing three times with PBS and resuspended in 0.5 ml media for mass spectrometric analysis. For a 50 ul cell sample, 20 ~.1 internal standard solution (40 ~eM verapamil, MW:454) and 5 ~Cl glycerol was added, and 4 ul of this mixture was used for FAB-MS. Several positive ion spectra were recorded in the mass range m/z 750-100 at a mass resolution of 1000, and a scan speed of 2 second/decade.
For mibefradil, m/z 496 was the dominant ion (M+H)' accompanied with a less intense sodiated molecular ion m/z 518. The concentrations of the mibefradil and hydrolyzed mibefradil were obtained by comparing the intensities of m/z 496 and 424 were to the intensity of m/z 455. For calibration, a standard solution of 50 ACM
drug was subjected to mass spectronnetric analysis.
Separation of cytosolic and membrane components -After washing out mibefradil from 'the bath solution, the cells were collected and the membranes were broken down by vortexing the cells in a solutican containing 5% acetic acid/CH3CN. The mixture was then spun and the.supernatant collected and defined as non-membrane associated components. Pellets were re-suspended in 5 x volume of NaOH (10 N) :methanol (1:7) solution at 37°C for 5 min.
The mixture was neutralized with 0.5 M HC1 and spun down. -The remaining pellet and the supernatant were collected separately.
Statistic s- All data is pre:~ented as mean ~ s.d.
and the student's t-test was used to calculate p values where given.
EXAMPhE I
Identification and Cloning of a Pancreatic T-type Calcium Channel The subject invention provides a cDNA encoding a T-type Ca2' channel al subunit derived from the rat insulin secreting cell line, I:NS-1, which has been identified and sequenced. The sequence of the cDNA
indicates a protein composed of 2288 amino acids (SEQ ID
N0:2), sharing 96.3% identity to the neuronal T-type Ca2+
channel al subunit (a1G) . The tran:~membrane domains of the protein are highly conserved but the isoform contains three distinct regions as well as 10 single amino acid substitutions in other regions. Sequencing rat genomic DNA revealed that this is an alternative splice isoform of alG. Using specific primers and reverse transcription polymerase chain reaction (RT-PCR) it was demonstrated that both splice variants are expressed in rat islets.
The isoform deduced from INS-1 was alsa expressed in brain, neonatal heart and kidney. Functional expression of this a1G isoform in Xenopus oocytes generated low-voltage activated Caz+ current~~. These results provide the molecular biological basis for studies of function of WO 00!15845 PCT/US99l19675 _ 52 _ T-type Ca2' channels in ~i-cells where these channels play critical roles in diabetes.
The cloning and tissue distribution of an isoform of the T-type Ca2' channel (a1G-INS) derived from the rat insulin-secreting cell line, INS-l (Asfari et al. 1992), -is described further below.
Based on the conserved amino acid sequence comprising the six transmembrane segments in repeat III
of the previously cloned al-subunit (Stea et al. 1995), degenerate primers were designed to deduce the cDNA
sequence of voltage-dependent Ca2+ channel from INS-1 which expresses a high level of T-type Ca2' current (Bhattacharjee et al. 1997). A 433 base pair (bp) DNA
fragment was obtained. The rapid amplification of cDNA
ends {RACE) strategy was then used to obtain the entire sequence of the channel. The full length cDNA (SEQ TD
N0:1) encodes a protein containing 2288 amino acids (SEQ
ID N0:2).
The T-type Caz' channel gene cleduced from (3-cells shares 96.30 amino acid identity with alG, the neuronal isoform of T-type Ca2+ channel (Perez-Reyes et al. 1998).
The four intramolecular homologous transmembrane domains of ~i-cell T-type Ca2+ channel a~ su:bunit are identical (except glycine 1667) to alG, with each repeat containing six putative membrane-spanning regions (S1-S6) and a pore-forming region (P-loop). The other highly conserved region is located at the intracellular loop between repeat I and II, where a section of histidi.ne-rich chain is present in the (3-cell derived T-type Ca2+ channel gene as well as in neuronal and cardiac T-type Caz+ channel genes. This structure in the loopl_IZ has not been observed in the protein sequences of known high voltage activated Ca2' channels.

In addition to the single amino acids that differ from alG, the T-type Ca2+ channel gene derived from ~i-cells contains three unique regions that differ from the amino acid sequence of alG. These regions are located at the N-terminal amino acids (aal-34 of SEQ ID N0:2), -intracellular loop III_III (aa971-994 of SEQ ID NO: 2 ) and intracellular loop LIIZ_IV (aa1570-:1588 of SEQ ID NO: 2 ) .
Although the amino acid sequence of the deduced channel is entirely different from. the a1G in the N-terminal region (aal-34 of SEQ ID NO:2), the nucleotide sequences at this region are almost identical except for 4 single nucleotide insertions which are shown in Fig.
1A. These four single nucleotide insertions determine a different start codon as well as those of the amino acid sequences.
To resolve the relationship between the T-type Ca2+
channel isoform deduced from INS-1 and alG, a section of Sprague-Dawley rat genomic DNA sequence containing the introns and exons between 4845 and 5256 was identified.
As shown in Fig. 1B, an exon was found that encodes the a1G fragment SKEKQMA (SEQ TD N0:5) as well as an exon that encodes fragment 4869-4922 of the INS-1 variant. This region also contains 8.5 kilobases (kb) of intron sequence. Thus, the T-type Ca2' channel al subunit cloned from INS-1 and a1G are alternative splice isoforms of the same gene.
The genomic DNA sequence wasalso used to examine the two nucleotide discrepancy between the a1G cDNA and the isoform cloned from INS-1. The data show that the genomic nucleotide sequence encoding amino acid 1667 is GGC (glycine), which is the same as the cDNA of al subunit cloned from INS-1 and the corresponding residue in alH, but is different from a1G (GCG, alanine) . Also of note, there are nine additional single amino acid substitutions WO 00/15845 PCT/US99I-19.67-5 in the isoform deduced from INS-1 as compared to the alG.
Six correspond to the amino acids found in the analogous position of alH: cysteine 1088, glycine 1667, alanine 1700, aspartic acid 1735, threonine 1812, and leucine 1813.
In regard to tissue distribution of T-type Ca2+
channels deduced from (3-cells and from neurons, expression of the ~i-cell T-type Ca2+ channel was found in rat brain, heart and kidney, but eras absent from liver.
Both a1G and the splice form were detected in rat islets and INS-1 cell preparations using RT-PCR. No a1H was detected.
Functional expression of the T-type Ca2+ channels deduced from (3-cells has been conducted in Xenopus oocytes using a double-electrode voltage-clamp method. In a solution containing 40 mM Ca2+, a family of current traces representing T-type Ca2+ current characteristics were obtained (Fig. 2A). The current slowly activated at -40 mV and peaked at -10 mV. The analysis of time constants of activation and inactivation are shown in Fig. 2B. The voltage-dependent activation {Fig. 2C) and steady-state inactivation {Fig. 2I~) uvere fitted with Boltzmann equation. The calculated Vl~z"s were -23.8 mV
and -45.6 mV for activation and inactivation, respectively; and k 's were 5.3 and -6.0 for activation and inactivation, respectively.
The nucleotide cDNA (SEQ ID N0:1) and amino acid (SEQ ID N0:2) sequences of rat pancreatic T-type calcium channel were determined. SEQ ID N0:3 is the nucleotide sequence beyond the coding region, while SEQ ID N0:4 includes SEQ ID N0:2.

EXAMPLE II
Characterization of the T type Calcium Channel in Relation to D:Labetes Glucose stimulated insulin release is Caz' dependent process, involving closure of the ATP-sensitive potassium-channels, depolarization and opening of the voltage-dependent Ca2' channels. At glucose concentrations below 3 mM; which do not elicit insulin secretion, [i-cells are electrically silent with a resting' membrane potential of L0 about -70 mV. Raising external glucose produces a slow depolarization, the extend dependent upon the glucose concentration. At glucose levels which elicit insulin release (>7 mM) depolarization is sufficient to reach the threshold potential (-50 mV) at which electrical activity is initiated.
A simple model for glucose-stimulated insulin secretion is summarized in Fig. 12. The resting membrane potential of (3-cells is principally determined by the activity of the K-ATP channel. When plasma glucose rises, its uptake and rate of metabolism by [i-cells are stimulated. As a consequence, the intracellular ATP (or ATP:ADP ratio) increases which leads to the closure of K-ATP channels and membrane depolarization. This results in the activation of voltage dependent Ca2~ channels (T-type and L-type) and the initiation of electrical activity. The increased calcium influx leads to a rise in [Ca2'~i and consequently insulin secretion.
Rat and human pancreatic (3-cells are equipped with L-type and T-type Ca2* channels. T'he physiological function of T-type Ca2' channels in. ~i-cells insulin-secretion has been demonstrated. These channels facilitate exocytosis by enhancing electrical activity in these cells. L-type and T-type Ca2+ channels, under normal conditions, work in concert promoting the rise in WO 00/15845 PCTlUS991t9675 [Ca2+~i during glucose-stimulated insulin secretion. In [i-cells, over-expressed T-type Ca2+ channels area at least in part, responsible for the hyper-responsiveness of insulin secretion to non-glucose depolarizing stimuli in GK rat, and in rat with NIDDM induced by neonatal -injection of streptozotocin. However, over-expressed T-type calcium channels over time will ultimately lead to an elevation of basal Caz' through its window current properties. Therefore, there is a dual effect of T-type Ca2' channels in [i-dells depending upon channel number and membrane potential.
Pharmacologically antagonizing T-type calcium channels is an appropriate treatment protocol for alleviating both insulin resistance and enhancement of insulin secretion in NIDDM patients.
NIDDM pathogenesis is complex: and the disease progression occurs in phases. An enhanced ~i-cell responsiveness provokes and initiates the disease process. It is unclear as to what the actual enhanced activity is and what the triggering mechanisms are for this first phase. It may be an increased secretory response or an increase in [i-cell mass. However, there is clearly an enhancement of [i-cell activity detected by both basal and postprandial elevated insulin levels denoted as hyperinsulinemia. Consequently, a resulting insulin resistance occurs, phase II, particularly in insulin responsive tissues (muscle, liver, kidney, fat) that function to reduce glucose levels in the blood. A
decrease in insulin sensitivity will account for an increase in blood glucose, causing the [3-cells to secrete even more insulin to compensate and because of this vicious cycle, full blown NTDDM, marked by an inevitable defect in insulin release, hyperglycemia and insulin WO 00/15845 PCT/US99/i9575 resistance, will characterize the final stage of the disease process.
Each phase of the disease may be characterized by an alteration in [Ca2+];, and each phase can be treated by a T-type calcium channel antagonist. The electrical (3-cell -is equipped with two types of voltage-dependent calcium channels, L-type and T-type calcium channels. L-type calcium channels, activated at high voltages, having Large unitary conductance, and dih.ydropyridine-sensitive, are considered the major pipeline for calcium influx into the ~i-cell (especially at high voltage depolarization).
T-type calcium channels, activated at low voltages, with small unitary conductance, and dihydropyridine-insensitive, are important for maintaining basal [Ca2']i (Fig. 8), as well as enhancing electrical activity during cell depolarization. T-type calcium channels normally facilitate insulin secretion in ~i-cells by enhancing cell electrical activity. This modulatory function of T-type calcium channels in insulin secretion is significant during phase I prior to onset of diabetes. Antagonizing these T-type calcium channels will decrease ~i-cell hyper-responsiveness and consequent hyperinsulinemia arresting the pathogenic pathways that lead to NIDDM.
If hyperinsulinemia and associated insulin resistance has already occurred, a T-type calcium channel Mocker is still the appropriate treatment protocol. The insulin responsive tissues, those that are primarily responsible for taking up glucose for re-establishing euglycemia, have elevated basal [Ca2']i during hyperinsulinemic conditions. Tndeed, it is the elevated basal [Ca2+]; that precipitates the decrease in insulin sensitivity of these tissues and it is now known that most of these insulin responsive tissues express T-type calcium channels. A T-type calcium channel blocker will reduce the basal [Ca2']i and alleviate the decreased insulin sensitivity.
Once NIDDM has manifested, it: is characterized by altered glucose metabolism, a result of abnormal glucose stimulus-secretion responsiveness of ~3-cells. (3-cell -desensitization to glucose is the principal secretory defect of NIDDM. L-type and T-type calcium channels, under normal conditions, work in concert promoting the rise in [Ca2~]i during glucose-stimulated insulin secretion. In NIDDM, this partnership is broken and the necessary rise in [Ca2+] i for insulin secretion is compromised.
The data herein indicates that L-type calcium channels are finely regulated by basal calcium levels {Figs. 9A-9D). A very small rise in basal calcium will substantially decrease the L-type calcium current and severely reduce the depolarization-induced rise in [Ca2+]i (Figs. 10 and 11). The data herein also suggests that T-type calcium channels are a primary regulator of resting basal [Ca2'] i in (3-cells. Furthermore, the negative feedback regulation of T-type calcium channels by elevated [Ca2+] i is absent (Figs . 9A-9D) . It is under circumstances of enhanced T-type calcium current activity as seen in the GK rat model of NTDDM and in the neonate streptozotocin-induced diabetes model, that basal [Ca2+]i is elevated, and a defect in the glucose-stimulated insulin secretion is observed. Simply reducing the basal calcium influx by pharmacological intervention, in situations of enhanced T-type calcium channel expression, 30 may reduce basal [Ca2'] i in (3-cells (Fig. 8) and alleviate the [Ca2~]i-induced inhibition of L-type calcium channels.
There is a clear link between. [Ca2'] i and diabetes.
A primary abnormality in [Ca2']i handling by cells is the defect initiating parallel impairments in insulin secretion and insulin action, as well as initiating diabetic complications. Consequent metabolic derangements may further aggravate alterations in [Ca2*]i homeostasis, creating a relentless cycle leading to 5 progressive deterioration in the overall health of the diabetic patient. Pharmacologica7_ agents that regulate [Ca2*]i homeostasis are thus appropriate therapeutic measures. The use of T-type calcium channel blockers will thus effectively treat and perhaps cure diabetes mellitus.
EXAMPLE II:I
Pharmacology of Mibef:cadil Action It has been shown that mibefx-adil has a potent inhibitory effect on T-type Ca2* current in vascular smooth muscle cells. The data hex-ein demonstrates that, in convention whole cell patch clamp configuration, mibefradil also blocks T-type Ca2* current in pancreatic ~i-cells. Mibefradil (1 ~.M) had been administered in the 20 recording chamber at time zero {F~.g. 13), the control (no drug) showed "run down". This figure shows that T-type Ca2* current is more sensitive to rnibefradil than the L-type Caz* current in pancreatic ~i-<:ells .
The blockade of T-type Ca2* channels in (3-cells with mibefradil is reversible. Fig. 19: demonstrates the reversibility of blockade of T-type Ca2* curx-ents by mibefradil. In these experiments, a very little volume of mibefradil or NiCl2 was delivered near the recording cell. The drug then diffused away from the cell. The 30 final concentration in the chamber was 1 nM. This experiment shows the inhibitory effect of mibefradil on T-type Ca2* current in pancreatic [3-cells results from reversible interaction between the: drug and the channel protein.

WO 00/15845 PCT/US99/19~75 In [i-cells, T-type Caz* channE~ls could mediate a small, but sustained, Ca2* influx by means of their unique "window" current at voltages near resting membrane potentials. Like other voltage-regulated channels, T-5 type Ca2* channels are opened and closed depending upon -the potentials across the cell membranes. This voltage dependency is illustrated in Fig. 15. The activation and inactivation curves represent the percentage of the channels in either open or closed states over a range of 10 voltages. Unlike most of the voltage-dependent Na*
channels or L-type Ca2* channels, t:he activation and inactivation curves of T-type Ca2* channels overlap at the certain range of low voltages (i.e. window).. In other words, there is a small portion of: T-type Ca2* channels 15 that stay in non-inactivated states in this voltage range. The data in Fig. 15 was obtained from experiments conducted under 10 mM external Ca2* condition, which shifted the window current about 1.0 mV toward positive voltage due to the surface charge effects of divalent 20 ions on the channels.
The existence of a window current provides a negative feedback regulation of [C'.a2*] i in (3-cells . When cells are under an unhealthy condition, they may be slightly depolarized to activate window current, which 25 elevates the basal [Ca2*]i to protect the cells from further Ca2* influx through the L-type Ca2* channels. This process is reversible if the membrane potential is reset to the normal resting potential (-70 mV).
30 Mibefradil regulates basal [Ca2*]; in pancreatic [i-cells:
The data herein demonstrates the roles of T-type calcium currents in modulating basal [Ca2*]a in INS-1 cells (Fig. 8). [Ca2*]i was directly measured by the ratio of fluorescence excitations at Ca2*-bound (380 nm) WO OU/15845 PCT/US99/i9675 -to unbound (340 nm), and then the ratio was converted to the calcium concentration. The bath solution contained mM NaCl, 4 mM KC1, 2 mM CaCl2, and 2 mM MgClz. In a single cell exhibiting fluctuating basal [Ca2']i with an 5 average value near 150 nM, administering 1 ~,M mibefradil -into the chamber immediately lowered the basal calcium.
This data shows the T=type calcium currents participate in regulating the mean basal [Ca2+]i in cultured ~3-cells:
10 Mibefradil regulates basal insulin secretion:
The activation of T-type Ca2' channel at low voltage near the resting membrane potential of pancreatic (3-cells suggests that the channels are responsible for the Ca2' influx required for insulin secretion under non-stimulus conditions. The NIT-1 cell line was chosen to demonstrate the effect of mibefradil on the basal insulin secretion. NIT-1 is a cell line derived from the (3-cell of non-obese-diabetic mouse. Thi;~ cell line expressed high levels of T-type Ca2' current. The data herein shows 20 that 5 uM mibefradil reduced the basal insulin secretion to less than 40% of control (Fig. 17), indicating this drug is able to lower the high ba:~al insulin secretion level seen during the earlier stage of NIDDM.
Spontaneous elevation of [Ca2+] i To demonstrate that T-type Ca2+ channels play an important role in calcium entry under non-stimulatory conditions, and therefore regulates basal [Ca2+];, spontaneous elevations of intracellular free calcium concentration was detected with the Fluo-3 AM fluorescent imaging method. NIT-1 cells were cultured in medium containing 3.3 mM glucose and preloaded with 2.5 ~M Fluo-3 AM. The numbers of spontaneous calcium elevated cells were counted and compared to, the total cells being used WO 00/15845 PCT/US99/~9675 - 62 - _ .
for a 10 minute observation period. l0 ~M NiClz inhibited 900 of spontaneous elevation of basal Ca2+.
The cellular mechanism of thE1 spontaneous elevation of intracellular Ca2+ was investig<~ted with the epi-fluorescence measurement method. Some INS-1 cells were -observed to exhibit transient spontaneous elevations of [Ca2*]i, "Calcium spikes", under non-stimulatory conditions. The Role of T-type Ca2* channels in this spontaneous process was examined as well. In a single cell with spontaneous calcium spike activity (Fig. 17), N,iCl2 (30 ~M) reduced the frequency of spontaneous calcium spikes immediately. This result s~~uggests that either the T-type Ca2' channels alone or together with the L-type Ca2+
channels are responsible for the transient spontaneous elevation of [Caz~]i, under conditions where na glucose is present. These spontaneous calcium spikes may contribute to basal insulin secretion and control of basal [Ca2+]i.
However, neither mibefradil nor NiClz exhibited their effect on basal [Caz+] i in all of t:he (3-cells. It was observed that only those cells which had relatively higher initial basal [Ca2+)i will r~sspond to the T-type Ca2+ channel antagonists (Fig. 18). Whereas those cells with lower initial basal [Ca2r]i had no or less response to the T type Caz' channel antagonists. This result indicates that T type Ca2+ channel antagonists may selectively act on the cells with high basal [Ca2+]i and bring it back to normal, by inhibiting the window current.
EXAMPLE IV
Action on Pancreatic ~-cells T type Ca2+ may play two pathological roles in NIDDM.
At the earlier. stage, the NIDDM patients exhibit WO 00/15$45 PCT/US99/'19675 hyperinsulinemia and ~i-cell hypere:xcitability. This may, at least in part, be due to increased activity of T type Ca2* channel in ~i-cells. At the more developed NIDDM
stage, over-expressed T type Ca2* channel and membrane depolarization resulted from reduced generation of ATP; -and may set up a window current in (3-cells that causes chronic elevation of basal Ca2* in the ~i-cells. The elevated basal Ca2* will reduce the L-type Ca2* activity and glucose induced insulin secretion.
It has been shown that mibefr<~dil prevented and reversed development of hyperinsulinemia in rat. This result indicates this drug is a valuable candidate for the treatment of earlier stage NIDDM or for preventing NIDDM in the potential patients.
A series of experiments were conducted with INS-1 cells to show that T type Ca2* facilitated insulin secretion by enhancing the general excitability of pancreatic a-cells. Particularly, activation of T type Ca2* channels will increase the firing frequency of the depolarizing spikes mediated by opening L type Ca2*
channels (Fig. 19A). Activation of T type Caz* channel will also decrease the time of devE:loping action potential elicited by up-threshold depolarizations (Fig.
198).
To further establish that T type Ca2* current enhances [3-cell excitability, 100 ~,cM NiCl2 was administered to effectively block 7.' type Ca2* channels.
In contrast to control experiments, NiCl2 caused a delay in the onset of an action potential. and a decrease in number of action potentials.
To directly demonstrate the role of T type Caz*
current in glucose-induced insulin secretion, INS-1 cells were incubated with 11.1 mM glucose: and variable concentrations of NiCl2, and insulin release was measured.

WO 00/15845 PCT/US99/19.675 - &4 -NiCl2 reduced insulin secretion in a dose-dependent manner (Fig. 20A). On the other hand, clonal insulin secreting cells (HIT-T15, which did not consistently exhibit T type Ca2+ current) were not affected by NiCl2 (Fig. 20B).
These results show that T type Ca2' channels play an important role in [3-cell excitability and antagonists of T type Ca2+ channels (such an NiClz;) will effectively reduce the excitability of [3-cells.
Although T type Ca2+ channels facilitate insulin secretion by enhancing general excitability of (3-cells, the function of T type Ca2+ channels is a doubled-edged sword. Under the conditian of over-expressed T type Ca2' channel in (3-cells, the function of the window current will become dominant and result in an elevation of basal Ca2+. High [Ca2'] i may cause impairment of insulin release by inactivating L type Ca2+ channels.
L-type Ca2+ channels are partially inactivated by [CaZ+1 i in non-stimulus condition in (3-cells:
Upon establishment of a whole-cell patch, within the first five minutes, the L type Ca2+ current "runs-up", as the magnitude of the peak current increases over time in INS-1 cells (Fig. 21). This phenornenon is a universal feature in these cells under the recording conditions used. The pipette solutions conta~Lned no ATP but did contain high cancentrat~.ons of the calcium chelating agents BAPTA and EGTA. When the pipette solution contained high Ca2', this run-up dof~s not occur. Tnstead, a rapid run down occurs. The "run-~up" phenomenon is likely due to calcium chelation in_~ide the cells. T type Ca2+ currents do not show this effect.

WO OOII5845 PCT/US99/19.675 _ 65 - _ Intracellular perfusion patch clamp experiments demonstrated that basal (Ca2*]i regulates L type Caz*
current amplitude in INS-1 cells:
Intracellular perfusion of a solution containing high Caz* {Fig. 9A) causes a substantial reduction in the -L type Caz* current. L type Ca2+ currents were elicited by a voltage step to +10 mV from a holding potential of -80 mV. The [Ca2*]i was measured directly using fura-2 ratiometric fluorescence. The effect of a high [Ca2+]
(272 nM) on the IV relatipnship i~~ shown in Fig. 9B.
Perfusing in high [Ca2*]i, substant.ially reduces the high voltage current component, but doe's not affect the low current component . The high [Ca2+I i caused a shi f t in peak current to negative voltages, and Ca2* currents were enhanced at negative voltages. This effect seemed to result in a potentiation of the T type Ca2+ current (Fig.
9D). Slow deactivating T type Ca2* currents showed a shift in activation upon perfusion of high [Ca2+] i. This may account for the shift in IV. Various concentrations of [Caz*] i regulated the activity of L type Ca2* channels (Fig. 9C) . Perfusing a low [Ca2*] i from an existing high [Ca2+]i {632 nM to 0 nM) caused an increase in the L type Caz* current over time, however pez-fusing in high [Ca2*] i (0 nM to 272 nM and 0 nM to 632 nNf) inhibits the L type Ca2* current over time. The level~~ of [Ca2*] i therefore have regulatory effects on both the L type Caz* current and T type Ca2* current, with [Ca2*pl having significant feedback regulation on the L type Ca2* current.
Effect of basal [Caz*] i on Ca2* influx:
The effect of basal [Ca2+] i on Caz* influx was examined using the Ca2* dye indicator fura-2 and fluorescence measurements. Voltage-dependent Ca2* influx in a single cell was obtained by perfusion of an WO 00/15845 PCTIUS991i9675 66 _ osmotically balanced solution containing 50 mM KCl into the recording chamber. Voltage-dependent increases in [Ca2*] i occur primarily through ni:Eedipine sensitive Ca2*
channels. The resting basal [Caz*]i in INS-1 cells was 5 approximately 60-80 nM under the experimental conditions.-CCa2+] ~ was determined by a standard curve obtained from a fura-2 calcium imaging kit (Molecule Probes). The empirical Kd obtained for calcium binding to fura-2 in the system was 296 ~ 20 nM. When basal [Ca2*]i remains low, 10 subsequent voltage stimulation with 50 KCl induces rapid and large calcium influx into the cell and these calcium changes are stereotyped upon repetitive stimulation when basal calcium is restored (Fig. 10). In this experiment, following the 50 KC1 depolarization, the cell was 15 repolarized by perfusion of the original 5 mM KC1 solution. After repolarization, basal [Ca2*] i slowly reset and then a second 50 KCl depolarization induced a similar [Ca2*]i transient. When the basal calcium is not allowed to reset, a defect in the second voltage induced 20 calcium transient occurs (Fig. 11). In this experiment, after repolarization, the second depolarization was applied before basal [Ca2*]i could return to its original value, and thus, the [Ca2*] ; transi.ent is substantially reduced. These findings suggest t:.hat basal [Ca2*] i plays 25 a prominent role in the regulatior~ of voltage dependent Caz* influx in INS-1 cells. There:Eore effectors of basal [Ca2*]I will have important impact on the amount of calcium that can enter the cell.
30 Streptozotocin induced high basal [Ca2*]i inhibits KC1 stimulated Caz* influx:
To reiterate the importance of basal [Ca2*] ~ on voltage stimulated Ca2* influx, ba:~al [Ca2*] i in INS-1 cells was artificially enhanced by pretreating the cells WO 00/15845 PCT/US99t19b~5 with the toxicant, streptozotocin. Though it is know that streptozotocin induces DNA strand breaks, it has also been shown to induce Ca2+ channel activity in cells. The data shows that pretreating cells with 5 mM
5 streptozotocin for 1 hour, followed by 3 hour recovery, -causes a two-fold increase in basal calcium (Fig. 22).
These cells when stimulated by 50 KCl had reduced calcium influx compared to control cells.
EXAMPLE V
Inhibition of T type Calcium Channel with Mibefradil Metabolite It has been shown that mibefradil (Ro 40-5967) exerts a selective inhibitory effect on T-type Ca2' currents; although at higher concentrations it can antagonize high voltage-activated Caz+ currents. The action of mibefradil on Ca2~ channels is use- and steady state-dependent and the binding site of mibefradil on L-type Ca2+ channels is different from that of 20 dihydropyridines. By using conventional whole-cell and perforated patch-clamp, mibefradil is shown to have an inhibitory effect on both T- and :L-type Ca2' currents in insulin-secreting cells. However, the effect on L-type Caz' currents was time-dependent and poorly reversible in 25 perforated patch experiments. Using mass spectrometry it was demonstrated that mibefradil was trapped inside cells and furthermore, a metabolite of mibefradil was detected.
Intracellular application of this metabolite selectively blocked the L-type Ca2+ current whereas mibefradil exerted 30 no effect. This study shows that mibefradil permeates into cells and is hydrolyzed to a metabolite that blocks L-type Ca2+ channels specifically by acting at the inner side of the channel.

- 68 - _ Whole-cell patch clamp and a bath perfusion system were first used to establish the dose-dependent inhibition of mibefradil on both 'T- and L-types of Ca2' currents. The T-type Ca2; current was measured at -30 mV
when the membrane was held at -90 mV and the L-type current was measured at +20 mV when the membrane was held at -40 mV. The currents were measured twice at each concentration of mibefradil with 2 min in between measurements. The dose dependent :inhibition of T-type Ca2' 10 current is shown in Fig. 3A. The 50% inhibitory concentration (IC~o) was 865 nM. No time-dependent inhibition was observed. In contrast, the inhibition of L-type Ca2+ currents could not be :fitted with a one-to-one binding curve (Fig. 3B). Administration of 1 ~M
15 mibefradil progressively reduced I~-type Ca2+ current up to 70% of the beginning amplitude after 10 minutes (n = 4), which indicated that a more complicated pharmacological mechanism was involved in the action of mibefradil on the L-type Ca2' currents .
20 A drug diffusing system was then used to test the reversibility of the antagonism of: T- and L-type Ca2+
currents by mibefradil. Small volumes (approximately 2 ~.1} of drugs were delivered in close proximity to the recording cell with a quartz capillary positioned by a 25 micromanipulator. After administr2~tion, drugs diffused throughout the entire recording criamber containing 2 ml of bath solution. This drug diffu~;ing system was used to test the reversibility of 30 ACM of' NiCl2 on the T-type Ca2+ currents (Fig. 4). The amplitude of T-type current 30 was rapidly reduced to 40% and gradually returned to 80%
of the initial level within 3 minutes. Using this system, it was found that the inhibition of mibefradil on the T-type Ca2+ current was clearly reversible. In contrast, WO 00/15845 PCTlUS99f19675 the inhibition of the L-type Caz+ current was poorly reversible (Fig. 4).
The poor reversibility and mime-dependent inhibition of the L-type Ca2' current by mibefradil suggested that 5 this drug may have an accumulaticm effect over time. This hypothesis was tested by applying a very low dose of mibefradil on cells and recording the L-type Ca2+ currents for a long time in the perforated patch-clamp configuration. As spawn in Fig. ~>A, after 25 minutes of 10 10 nM mibefradil administration, the relative currents were reduced to 70%, whereas the currents remained unchanged for control patches. Incubation of cells with nM mibefradil for two hours resulted in further reduction of current densities as recorded by perforated 15 patches (Fig. 5B). At a concentration of 10 nM, mibefradil exhibited no long-term effect on the T-type Ca2+ current .
To test the hypothesis that mibefradil may permeate through the cell membrane to the cytoplasm and be trapped 20 inside cells, the presence of mib~efradil was examined in cells pre-incubated with 20 ~.M of mibefradil using mass spectrometry. After 3 washes, mibefradil (peaked at 496 MW) was still detected in cells (Fig. 6B). The concentration of intracellular mibefradil after one 25 minute incubation was 3.18 ~ 0.78 ~.M (n = 3). The localization of mibefradil in cel:Ls was examined by measuring the concentration of mibefradil in the pellets and supernatants after lysis of the cells. Most of the mibefradil (92%) was detected in t:he supernatants and 0%
30 was found in the pellets after wa:~hing cells with methanol, indicating that mibefractil was trapped in the cytoplasm. In addition, a peak (MGiI = 423) was detected which represented a hydrolyzed metabolite of mibefradil, Des-methoxyacetyl mibefradil (dm-rriibefradil), which is a major metabolite as documented previously (Wiltshire et al. 1992). By varying the time of~pre-incubation, it was found that dm-mibefradil accumulated inside the cells in a time-dependent manner (Fig. 6A). This accumulation is consistent with the concept that dm-mibefradil has lower -membrane permeability than its precursor mibefradil.
Tt was then tested whether or not mibefradil or dm-mibefradil inhibits L- or T-type Caz+ currents from inside of cells. Both L- and T-type currents were 10 measured in the whole-cell patch clamp configuration when 1 ~,M of mibefradil or dm-mibefradil was included in the pipette solution. As shown in Figs. 7A and 7B, intracellular application of 1 uM mibefradil did not have inhibitory effects on either L-type or T-type Ca2 15 currents, whereas the same concentration of dm-mibefradil specifically blocked the L-type Ca2' current.. As the bath solution contained no drug in this series of experiments, the inhibitory effect of dm-mibef:radil is considered to be acting on the inside domain of L-type Ca~+ channels.
20 The inhibitory effect of dm-mibefradil on T-type Ca2+
currents was similar to the effect of mibefradil when it was applied in the bath solution, suggesting that the methoxyacetyl group of mibefradil does not play a key role in binding to the extracellu:lar receptor site of 25 T-type Ca2+ channel protein. However, this methoxyacetyl group is necessary for blocking L-type Ca2+ channel from the inside of cells, indicating a modification in the methoxyacetyl group of mibefradil can result in a more selective antagonist of T-type Ca''' channels.

EXAMPLE V:I
LVA Caz* Current Mediates Cytokine-Induced Pancreatic ~-cel,1 Death Insulin-dependent diabetes mellitus is characterized by the selective destruction of pancreatic [3-cells. ' Chronic treatment with cytokines induced a low voltage-activated (LVA) Ca2* current in mouse ~i-cells . The concomitant increase in the basal cytoplasmic free Ca2*
concentration ( [Caz+] i) was associated with DNA
fragmentation and cell death. Antagonists of LVA Ca2*
channels prevented this elevation of basal [Ca2*]i and DNA
fragmentation, and reduced the percentage of cell death.
Exposure to cytokines did not affect the profile of Ca2*
currents or basal [Ca2*] i in glucac~on-secreting a-cells .
An increased Ca2* signal through LVA Ca2* channels may thus be a key feature in cytokine-induced (3-cell destruction.
The effects of chronic cytok:ine treatment on the voltage-sensitive Ca2* currents in primary cultured mouse islet cells was examined. After treatment with IL-1[i (25 U/ml) and IFNY (300 U/ml) for 6 h, an LVA Caz* current was induced in these cells (Fig. 23A). This current was present in 48% of cytokine-treate<3 mouse islet cells. No such current was observed when the cells were treated with either IL-1[i or IFNy alone. Experiments were conducted at different times recording LVA Ca2* currents induced by cytokines, and the results indicate that no further increase in current densii~y occurs even after treatment for 48 h. This LVA current has not been observed in non-treated cells. The steady state inactivation curve of the cytokine~-induced LVA Caz*
currents displayed a low voltage property (Fig. 23E.) similar to the inactivation curve of the LVA currents in NOD mouse islets cells. This current was also blocked by NiCl2 (10 ~,M; n = 4; Fig. 23F). It has been reported that low concentration of NiCl2 selectively block LVA current, a profound increase in Ca2* current density was observed over the voltages between -20 and 20 mV. These high voltage-activated Ca2* currents are nifedipine sensitive currents (completely blocked by 10 ~.M nifedipine), and the increase in this current density is similar to the increased L type Caz* current density observed after treatment of [3-cells with serum from IDDM patients.
As a-cells are more resistant to the toxic effects of cytokines than (3-cells, the effects of cytokines on the Ca2* currents in a glucagon-secreting cell line (a-TC1) was also examined. This cell line, like a-cells, is more resistant to the cytotoxic effect of cytokines.
Treatment of a-TCl cells with IL-1(3 anal IFNy failed to induce LVA Ca2* currents and did not alter the current density (Figs. 23C and 23D). Therefore, the induction of LVA Caz* currents and increased Ca2* current density observed after chronic treatment iNith cytokines showed specificity for [3-cells.
LVA Ca2* channels are activated at low membrane potentials. This unique feature may allow then to regular [Ca2*] i under nonstimulatory conditions . Indeed, basal [Ca2*]i in cytokine-treated cells was approximately 3-fald higher than in nontreated cells (Fig. 24A). This increase in basal [Ca2+] i was bloc7~:ed by NiClz (10 ~,M) , but not by the L type Caz* channel antagonist, nifedipine (10 ~M). Cytokines failed to increase basal [Caz*]i in a-TC1 cells (Fig. 24B) . These results :suggest that Ca2* influx through LVA Ca2* channels is responsible for the cytokine-induced elevation in basal [Ca2*] i in (3-cells.
Tt has been shown that cytokines induce apoptosis in human pancreatic islet cells. Apoptosis is also the mode of cell death in the development of IDDM in the NOD mouse and in multiple low dose streptozotocin-induced TDDM in the mouse, and is involved in ~S-cell destruction. As a marker of apoptosis, DNA fragmentation has been reported to precede (3-cell lysis .
~i-TC3 cells, a mouse ~3-cell 7_ine, were used to demonstrate the role of LVA Ca2' channels in cytokine- -mediated DNA fragmentation. The LVA Ca2+ current density was first examined before and after cytokine treatment.
The LVA Ca2+ current (at V", _ -30 mM) in ~i-TC3 cells was increased from 1.86 ~ 0.33 (pA/pF; n = 30) to 3.45 ~ 0.47 (pA/pF; n = 10) after treatment with cytokines (25 U/ml IL-1~3, 100 U/ml IFNy, and 100 U/ml. TNFa) for 25 h. This indicates that the LVA Ca2' current: in (3-TC3 cells is regulated by cytokines, as seen in mouse islet cells. As shown in Fig. 24, cytokine-induced: DNA fragmentation displayed a ladder pattern of olig~onucleosomal fragments.
The three LVA Ca2' channel blocker~~, NiCl2, amiloride, and mibefradil, all independently prevented cytokine-induced DNA fragmentation. Tn contrast, n.ifedipine had not inhibitory effect on DNA fragmentation induced by cytokines. This experiment has been repeated in (3-TC3 cells (n = 2) as well as in NIT-1 cells (n = 3), a j3-cell line derived from NOD mice, and the same results were obtained.
The function of LVA Ca2+ channels in cytokine-mediated cell death in ~3-TC3 cells was then examined.
Many cells died when the medium contained 25 U/ml IL-1(3, 100 U/ml IFNy, and 100 U/ml TNFa; however, NiCl2 (20 ~M) effectively reduced the ~i-cell killing potency of cytokines in both a time- and dose-dependent manner (Figs. 25A and 25B, respectively). Tn contrast, nifedipine did not exhibit a protective effect. Similar results were obtained from an experiment conducted in NIT-1 cells with mibefradil, which also reduced (3-cell death induced by cytokines. These results demonstrate WO 00/15845 PCT/US991r9f75 _ 74 _ _ that LVA Ca2' channels enhance the vulnerability of (3-cells to the cytotoxic effects of cytokines.
Although preferred embodiments have been depicted 5 and described in detail herein, it will be apparent to -those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within 10 the scope of the invention as defined in the claims which follow.

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WO 00/15845 _ 1 _ PCTIUS99/19675 SEQUENCE LISTING
DNASISr DNA Translation IT-INSj File Name : T-INS
Range : 2 - 7286 Mode : No_:.al Codon Table : Universal ra t ~
5t y 5 ' GAG CTG AGC TGA ACT GGC CCT CCT GC-G (~C TCA G::_3 AGC 'iY:T CTA GAG CCC CCC

E L S , -T _ _ _ 64 73 82 91 100 7.09 ACA TGC TCC CCC ACC (N.; G'PC CCC CGG TTG CGT GAG GAC

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- _ d 118 127 136 145 154 163 s~ ~.
CTC CG: r5o~y T

. Q s~
CG CCC CTC TTC CGA CCC CCC GGG GCC CCG GCT GGC CAG AGG ~,,~
ATG G.AC
_ __ __ __ _ _ _ ___ ___ ___ -L F G P P G A P A G Q R M D
~' ~d G.~G GAG GAG GaT GGA GCG 'GG' GCC GAG C.=.G TCG G~1 CAG
CCC CGT AGC TT'C ACG
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G A G A 'E E S ~G . Q P R S ~F T

CAG CTC AAC GAC CTG TCC GG~v Cri.C G~.v GGC CGG CAG Gv~G
CCG GGG TCG ACG GnA

Q L N D L S G A G -G Ft ,~ _G rp~ - ~ _S -T _~_ RAG G.~C CCG GGC AGC GCG GAC TCC GAG GCG GAG Gt;,G CTG
CCG TAC CCG GCG CTA

K D P G S A D S E A Er G wL -P Y P A L

G.:.C CCG GTG GTT TTC TTC TAC TTG AG.:. CAG GAC Ac,3C
CGC CCG CGG AG:. TGG TGT

A P V V F F Y L~ rS 'Q 'D J5 'R P -R S W C

388 397 406 4:L5 424 433 CTC CGC ACG Cs"TC 'mT AAC CCG TGG TI'C GAG CG'.~ G'L'C
AGT ATG CIG GTC ATT CTT

L R T V C N P W .. F~ E ~R V S _li 'I~ -V, _Ir ~L

CTC AAC 1GT GTG ACT CTG GGT AiG TTC AGG CCG TGT GAG GAC
ATT CiCC TGT GAC

L N C V ~T 'L G M' F R P C- ~E D T A- C -D

TCC CAG CGC TGC CGG ATC (:TG CAG G:.C TTC GAT GAC TTC
ATC TTT GCC TTC TTT
-.

5'77 ' 586 595 GCT GTG GAA ATG GTG GTG AAG ATG CyTG GCC TTG Gtri. ATC
TIT GuG AAG AAA TGT

A V E M -~ _ _ _ _ _M _ A -~ t; T F ~G K K C

- 604 613 622 6:31 640 649 TAC CTG GGA GAC ACT TGG AAC CGG CIT GAC TIT TSCC ATT
GTC ATT GCA GGG ATG
_ -_T_ _~ _~ _R _~ _D _F_ _~1 _ _~_ _V _Z _A_ _G .M
D
Y L G

WO 00/15845 2 _ PCT/US99/l9575 DI~1SIS~ DNA Translation ~T-INS]

CTG G? G '.~~T TCG C?G GaC CTG CAG AAC GTC AGC TTC 'IBC GCs: GtC AG~v AC.3, GTC
L E Y S L _D -~ -Q_ _N _V _ _ _ _ .. _ V _R _ _ 722 721 730 7:39 748 ~ 757 CGT GTG C iG CGA CCG CTC AGG GCC ATF A.~C CGG G'.CG CCC AGC ATG CG.: A2T CTC
R V L R -p- L R- A I_ -N _R.. _ ~ _g _ _ GTC ACA TTA Cite CTG GAC ACC TTG CCT ATG CTG GGC. A.,C G2'C CTG C'IG C:'C TGT
V T L L L -D T L P M -L G- N V L L -L- C
820 829 838 8r17 856 865 TI'C TTC GTC TTT TTC ATC T iT G~. ATC G'ZG GGC GTC CAG CT3 TGG GCa G~ CIG
F F V F -F -I F~ G -I V G V Q L- ~W A G L
874 $83 892 901. 910 919 CTT CGC AAC CGA TGC TTC CTG CCC GAG ~'C TTC ACri. CTC CCC CTG AGC GTG GAC
L R N R C
928 937 . 946 9-°i5 964 973 CTG GAG CCT TAT TAC CAG ACA GAG A.~T GAG GAC GAG AGC CCC TTC ATC T~~: TCT
L E P ~ Y Y Q- T E -N -E- D -E: S p ~ F "__ _C_ _S_ CAG CCT CGG GAG AAT GGC ATG AGA TCC TvC AGG AGT GT'G CCC ACA CTG CGT GGG
Q P R E- N G- M R -S - -Ft S~ V P T L R G

GAA GGC ~T GGT GGC CCA CCC TGC AGT CTG GAC TAT G.~.G ACC TAT AAC AGT TCC
E G G G G -p -p C S L D Y' E T Y -N S S
1090 1099 1108 111,7 ~ 1I26 1135 AG.~. AAC ACC ACC TGT GTC AAC TGG AAC CAG TAC TA.T ACC AaC TGC TCT GCG GGC
S N T T C V- N W .. N- Q Y Y T N -C S A -6 1144 . 1153 1162 1271 1180 1189 E H N -P F 1C G A- ___ _H _ _ _~ _ __ _ _ _Y _A _~_ 1198 1207 I2I6 1225 ' 1234 1243 ATC GCC A.TC TTC CAG GTR ATC ACA CTG GAG GGC TGG GTC GAC ATC ATG TAC TTC
I A I F Q ~' -__ _T _~ _E _~_ -W' _v _p =_ _M_ _Y- _F

GTA ATG G1C GCT CsIC TCC TTC TAC AAC TTC ATC TAC TTC ATT CIT CTC A'IC ATC
V M D A- H S -F- Y N F I Y' F I L L I- I-1306 ~ I3I5 1324 1333 1342 1351 GTG:GGC TCC TTC TTC ATG ATC AAC Ct'G 2GC C2G GTG GTG ATT G.C ACG CAG TTC
V G . S F- F M , I N I, 'C -I,_ -V_ V -__ -A -T "Q F

WO 00115$45 _ 3 _ PCT/US99/I9675 DNASIS DNA Translation [T-INS]
1360 1369 1378 1:387 1396 1405 TCC GAG ACC AAA CAG CGG GaG AGT CAG C2G ATG <:GG GAG CAG CGT GTA CGA TTC
~ -K_ _Q_ _R _~ _S _Q_ _~ _~_ ._R _E _Q- _~ V_ _R_ _ 1414 1423 1432, I~I41 1450 1459 CTG TCC ACT GCT AGC ACC CTG GCA ACS TTC TCT C~ CCA GGC 'AGC TGC TAT GAG
L S N -A -S_ _~ _~ _A_ _S _~_ _S_ _.E_ _p _G _ _ _C_ _Y

C?G G'TA CTC AAG TAC CTG C'TG TAC ATC CTC Ci:A Fu~.~ Cu1 GCC CC'ui' AGG C"FV~
GCC
E L L K y' L V Y -I L R .K -A A R R L A

CAG GTC TCT AG~v Cn.T ATA G.;aC C'TG CGG Cn.~T C~v C:2G CTC: AGC AGC CCA C"TG
GCC
Q . V S -R A I G , ~V R A G -L ' _ S Sw P _V A

CGT AGT GGG C,~G GaG CCC CAG CCC AGT GGC AGC TGC ACT CGC TC.~ CAC CGT CGT
R S G Q~ E ~p Q p S G 'S C T ~R S -H R -R

CTG TCT GIC CAC C.~C CTG GTC C.AC CAC CAT CAC CAC CAC CAT CAC CAC TAC CAC
L S V H~ H ~L- -H_ _H _ _ , _~_ -CTG GGT AAT GGG ACG CTC AGA GTT CCC CGG GCC FJ;ri. CCA CAG ATC CAG CSC AGG
L G N- G -T L ~R A p~ Ft _A _ S- p -g ___ Q D R
1738 1747 1756 1765 17?4 1783 GAT GCC A~.T C~,:~G TCT CGC CGG CTC A2G CTA CCA Cc:.F1 CCC TCT ACA CCC ACT CCC
D A N G S- R -R L- M L 'p_ _; _ _g _S_ _ _p - _p_ 1792 1801 1810 18:L9 1828 1837 TCT G~vG Gv~C CCT CCG.AGG GGT GCG Gr~G TCT GTA CAC AGC TTC TAC CAT GCT G?.C
S G G ' P~ p R G A .. E S V H S FV Y -H -A D
1846 ~ 1855 1864 18',T3 1882 1891 Tu'. CAC TTG GAG CCA GTC CGT T~uC CAG GCA CCC CC:T CCC AGA TGC CCA TCG GAG
C H L E -p- V R -C 'Q -A_ 1900 1909 1918 19:!7 1936 1945 GCA TCT GGT AGG ACT GTG GGT AGT GGG AP.G GTG TAC CCC ACT GTG CAT ACC AGC
A S G Ft T V G S G ~ K V 5~ p 1954 1963 1972 l9FIl 1990 1999 CCT CC.~ CCA GAG ATA CTG I~AG GAT AA.~I GCA CTA G'I'G GAG GTG GCC CCC AGC CCT
P p p E -~_ _L_ _K_ -D -~ A wL V E V -A - _ _ _ 2008 2017 2026 2035 ~ ' 2044 2053 GGG CCC CCC ACC GTC ACC AGC T~'C AAC ATC CCA CC:T GGG CCC TTC AGC TCC ATG
G p p -T -I. T -Sy 'F -N_ ___ ..p ..p' _G_ _p _F _S_ -S M

WO 00/15845 _ 4 _ PCT/LJS99/19675 DN~rSrS DNA Traaslatioa [T-=NS7 2062 2071 2b80 2;089 2098 2107 CAC AAG C'IC CTG CMG ACA C~.G AC'T ACG GGA Cri.C TGC CAT AGC TCC TGC AAA ATC
H K L L E rT Q S T G A C H S S C K ~I

TCC AGC CCT TGC TCC A~G GCA GAC AGT G~vA G:.C TGC GGG CCG GAC AGT TuT CCC
S S p 'C- -S_ '~ _A _D _S_ _G _~_ _ _ _ _ _ TAC TGT GCC CGG ACA GGA GCA GGA GAG CCA CnG TCC G.~T GnC CiaT G'T_"~' ATG CCT
Y C A R T G A G E p E S A -D rFI _V M - _ ~C TCA GAC AGC GAG Cn.'"T GTG TAT GAG TiC F.CA ~Cr'1G CuIC Cn.'"T CAG CAC AGT
GAC
D S D S- E~ A V Y E F T -Q 'D _A -Q h S D
2278 2287 2296 2.305 2314 2323 CTC CGG GAT CCC CAC AGC CGG CGG CG.~ CAG CGG .AGC CTG GGC CCA GAT GCA G.~G
L R D p -H S R R -R -(~ R ._S_ -~ _G_ _P_ _ _A
2332 2341 2350 2:359 2368 2377 CCT AGT TCT GTG CTG GCT TTC Tu~G AGG CTG ATC 'rGT Cu'~C ACA TTC CGG RAG ATC
P S S 'V L' ' A F W R L -I ~ C 'D T F R K I

GTA GAT AGC AAA TAC TTT GGC CGG GC.A ATC ATG ATC GCC A'IC C'IG GTC AAT ACA
V D S K y 'F' -G_ _ _G_ ___ _M .__ _A _~_ ._~ .. _H

CTC AGC ATG GGC ATC GAG TAC C'AC GAG CAG CCC (sAG GAG CTC ACC AAC GCC CTG
L S M G T ~' ~Y H E Q P . E~ -E Lr T N A ~L
2494 2503 2512 .2°.i21 2530 2539 GAA ATC AGC AAC ATC GTC TTC ACC AGC CTC TTC t~CC TTG GAG A2G CTG CTG AAA
E I S N I V- F Tr..'S L F A . -L -E- MT L _L -FC
2548 2557 2566 2:175 2584 ' 2593 C'1G CTT GTC TAC GGT CCC TTT GGC. TAC ATT AAG AAT CCC TAC ABC ATC TTT GAT
L L V Y G P -F ~G Y- I K ,N P Ya N I F~ Dr 2602 2611 2620 ' 2Ei29 2638 2647 GGT GTC ATT GTG GTC ATC AGT GTG TGG GAG ATT G"IG GGC CAG CAG GGA GGT GGC
G V I V V = -gw -V

L S V L R T -F R L M' R _,V _L _K _L _V _R_ _~_ 2710 . 2719 2728 2T37 2746 2755 CIG-_ CCG GCC C2~ -CAG CGC CAG CTC GTG G'TG CTC F.TG AAG ACC AZG Cz~C AAC GTG
L P A -L" -Q- _R_ _Q_ _~ -V_ v _~ _.M _K -T -M -D -N V

WO 00/15845 _ 5 _ PCTlUS99/i9675 DNASI$ DNA Transl.atioz~ (T-INSI
2764 2773 2782' 2791 2800 2809 GCC ACC TTC TGC ATG CTC CTC ATG CTG TTC ATC TTC ATC TTC AGC ATC CTG GGC
A T F- C M I, 2818 2827 ~ 2836 284 5 2854 2863 ATG CAT CTC '!TT G"~T TGC A? G TTC GC.'a TCT CAA Ct;~:, GAfi GGG GAC ACG TTG
CCA
M H L F' G -C
2872 2881 2890 2899 ~ 2908 2917 GAC CGG AAG AeIT T7:C GAC TCC CTG CrC TCv Cri.C ATC GTC ACT GTC TTT CMG AfiT
D R K N F
2926 2935 2944 2.'353 2962 2971 CTG ACT . CAG GAA GAC TGG AAT Arl~. GTC CTC TAC i4AC Gu~C ATG G~.C TCC ACA TCG
L T Q E D W N K V L Y N G M A S T S

TCT TGG GCT GCT CTT TAC TTC ATC GCC CTC ATG ACT TTT GGC A.~.C TAT GTG CTC
S W A- A -L 'Y- _ _ _ _A _~ _M .. _F_ _ , _~ -Y V L
3034 3043 3052 3061' 3070 3079 TTT A.~1C CTG CTG GTG GCC ATT CTT G'-G G1A GGA 7:TC CAG GCA GAG GAA ATC GGC
F N L L ~V -A' -I_ _~
3088 3097 3106 31.15 3124 3133 AAA CGG GAA GAfi GCG AGT GGA CAG TTA AGC TGT ATT CAG CTG CCT GTC AAC TCT
K A E D A S' G _Q _I, S 'C
3142 3151 3160 ' 3169 3178 318?
CAG GGG IsGA GAT GCC ACC AAG TCT GAG TCA GAG CCT GAT TTC TIT TCG CCC AGT
Q G G D A

GTG GAT GV~T GAT GGG GAC AGA AAG AAG CGC TTG GCC CTG GTG GCT TTG GGA GAA
V D G D ~ -D- -R" -~ ._K _R_ _~_ _A_ _~_ _V A _T~ _G -E

CAC GCG GAA CTA CGA AAG AGC CTT TTG CCA CCC CTC ATC ATC CAT ACG GCT GCG
H A E L -R~ K _ _ _~ _ _p _~_ _G __ __ _~.. -T _A -A
3304 3313 3322 ~ 33:31 . 3340 3349 AC.~ CCA ATG TCA CTA CCC AAG AGC T'C AGC ACA G3T GTG.GGG GAA GCA CTG GGC
T P M S' L -p _K _ _ _S- _ _ _ _ _ .,~ _ _ _ -Ly -G

TCT GGC TCfi CGA CGT ACC AGT AGC AGT GGG TCC Gc=T GAG CCT GGA GCT GCC CAC
S G S R R T S S -S~ G S J~ ~ -p_ G A -A _H

CAfi'GAG ATG AAA TCT CCG CCA AGT GCC CGC AGC TC:C.CCG CAC AGT CCC TGG AGT
H , E M K S p p ~g A -R- S _''., _p_ _~_ _S -p -W S

WO 00/15$45 _ 6 _ PCT/US99119675 Dt~ISIS DNA Translation [T-INSj GCG GCA AGC AGC 2GG ACC AGC AGG CGC T'C AGC :9,GG AAC AGC CTG GGC CGG G:C
A A S_ _ _ _W _T_ _S_ _R_ _R _ _ _ .. _ _ _ _~_ _G _ _ 3520 3529 3538 3'.47 3556 3565 CCC AGC CTA AAG CGG AGG AGC CCG AGC GGG GAG CGG ACv TCC CTG CTG TCT GGA
S L IC R _R_ _S_ _ _ 3574 3583 3592 3Ei01 3610 3619 GAG GiC CAG GAG AGT GAG GAT GAG GAG GAA AGT TCA GAA GAG GAC CGG GCC AGC
E G. Q E S Q D E E E S S E E D R ?. S
3628 3637 3646 3E>55 3664 3673 CCA GC_~ GGC AGT GAC C.~T CGC CAC AGG G""T TCC '!.'IG GAA CGT G..aG GCC A.~G
AGT
p A G~ S rD H R H R' G S .L E' R !E A K S

TCC TIT GAC CTG CCT GAC ACT CTG CAG G2G CCG GG~v CTG GEC CGC ACA GCC AGC
S F D L p _D -,~,_ _~ _ø v _p_ _,~ _~_ _H _R _T_ _A _ _ GGC CGG ACS TCT GCC TCT G.~G CAC G3e1 GAC TGT AAT GGC AAG TCG G~.~T TC_~1 GGG
G R S S A~ S~ E -H ~Q D C N G X ~S 'A yS 'G

CGT TTG GCC CGC ACC CTG AGG ACT GAT GAC CCC CAA CTG GAT GGG G.~T GAT GAC
R L . A _R _ , _ _ _~ _D _D _p_ _~ _~ _D _ _ _ _ _ AAT GAT GAG GGA A.iiT CTG AGC AAA GGG GAA CGC ATA CAA GCC.TGG GTC AGA TCC
N D E 'G N- L S K G ~E R I ~Q A 'W V- R~ S' 3898 3907 3916 3925 ' 3934 3943 CGG CTT CCT GCC TGT 'iGC CGA GAG CGA GaT TCC ThG TCG GCC TAT ATC TrT CCT
R L p A -c- -c_ _R. _E_.._R - -S 1iJ S Ar y -~_ 3952 3961 3970 39'79 3988 3997 CCT CAG TCa AGG TTT CGT CTC C'IG 2GT CAC CGG A'TC ATC ACC CAC AP.G ATG TTT
P Q S 'R F _R- L L C _~ wR " _ _~.. _x, _H -K 'M -F
4006 4015 4024 40:33 4042 4051 GAC CAT GTG GTC CTC GTC A2~ ATC TTC C2C AAC T«T A2G ACC ATC GCT ATG GAG
D H V V L V rI~ I F L ~N rC I T I~ A Mr E

CGC CCC AAA ATT' GAC CCC CAC AGC GCT GAG CGC A'.CC TTC CIG ACC Cite TCC AAC
R P K I D P rH rS A !E R ,[ F
4114 ~ 4123 4132 .4141 47.50 ~ 4159 TAC ATC TTC ACG GCA GTC TTT CTA GCT GAA ATG AC:A G2G AAG GTG GTG GCA CTG
Y I F ~T A ~V F L- A E- M T V K V ' V A L

WO 00/15845 _ ~ _ PCT/US99t19b75 DN?aS~S DNA Translation jT_INS]
4168 4177 4186 4:195 4204 4213 GGC TGG TGC TTT GGG GAG CAG GCC TAC CTG CCn.~AGC AGC TGG AAT G:G C3G GAC
G W C- F G E Q A Y L 'R 1 S S W' N~ V L D
4222 4231 4240 4;149 4258 4267 GGC TTG CIG GTG CTC ATC TCC GTC ATC GaC AT:. c~2G G:C TCC AT3 GTC TCC GAC
G L L V L- I S V I D I ~ L V ~S M V- S D

ACri. GGC ACC AAG ATC CTT GGC ATG CIG ACv GTG C:TG CGG CTG CTG CGG ACC G'TG
S G T K . -~' L _G~ _~ _ _V _.L_ _R _~ _ _ 4330 4339 4348 4:157 4360 4375 CGT CCA C"1'~ AGG GTC ATC AGC CGG GCC CAG GGA C."IG AAG CTG GTG GTA Crr~G ACT
R P L R V I S R A Q G L K L V V E T
4384 4393 4402 49'.11 4420 4429 CTG ATG TCG1 TCC CTC AAA CCC ATT GGC AAC ATT GTG GTC ATT ~C TGT GCC TTC
L M ~ S - _ _L
4438 4447 4456 44.65 4474 4483 TTC ATC ATT TTT GGA ATT CTC C,GG CiiG CAG CTC 'ITC A 3 GCrs AAG TTC TTC C=TG
FIIF'GIrLGVQL'FK-GKFrF'V

~T CMG C~"T GAG GAC ACC AC~G AAC ATC ACT AAC AA.A TCC GAC ZGC GCT GAG GCC
C Q G ~ -D -T -~ -~ _=r _ _ AGC 2'$C CCxA TGG GTC CGG CAC AAG TAC RAC TIT Gr~C A.'~C CTG GGC C:AG C'sCT
CiG
S Y R ~W V ~R H K Y N F :D- ~~

ATG TCC CTG TTT GTG CTG GCC TCC AAG GAT GGT 2~.7G GTT CSC ATC ATG TAT GAT
M S L F V L A S . K D G liJ V D I M Y- D

GGG CTG CaAT GCT GTG GGT GTG GAT CAG CAG CCC A'Z'C ATG AAC CAC AAC CCC TGG
G L D A -V- _G . _v_ _D _g _~ _~_ _.C_ _M _u ATG CTG CFA TAC TTC ATC TCC TTC CIC CTC ATC G'.DG GCC TTC TTr GTC CTG RAC
M L L Y F _I_ S- _~ _~ _~ ___ _ , _A_ _ _E _ _ _ 4762 47?1 4780 47Fi9 4798 4807 ATG TTT GTG GGC G2G GTG G2G Cx~IG AAC TTC C,AT AAG 2GC AGA CAG CAC C.AG GAG
M F V G -V V V E N F H _F: C R Q H -Q E

GAG.C,rIG C~P.G GCG AGG CGG CGT GAG GAG AAG CGA C'7:A. CGG AGG C2G GAG AAA AAG
E E E ~A R ~R 'R E E K ~R LY R R L E K K

WO OU/15845 _ g _ PCT/IJS991r9875 DNASiS~ DNA Traaslation [T-INS]
4870 4879 4888 48:97 4906 4915.
AGA AGG AAT CTA ATG TTG GAC GAT GTA ATT GCT TCC GGC AGC TCA GCC AGC Gw.~T
R R N -L- M L 'p - _ _V _z_ _~ _,,_ _G _S _S_ _A _S_ _~
4924 4933 4942 49liI 4960 4969 GCG TC-a G~. GCC CAG ZGC AAG CCC TAC TAC TL:T G:'.C TAC TCG AGF. TTC CGG CTC
A S E A Q _~_ 'K _p_ _Y_ _Y _S_ _I;_ _~ _S _ _ _ _ _L_ CIT GTC CAC CAC CTG TGT ACC AGC CAC TAC CTG G:~C CTC TTC ATC ACT G~~T GTC
L V E Fi L C T S FI Y L I) L F I T G Y
5032 5041 5050 50'.i9 5068 5077 A?'C GGG CTG P aC GTG (iTC ACT ATG GCC ATG G~1 CAT TAC CAG C=~G CCC CMG ATC
I G L N V V T M A- M E F~ y _Q 'Q' -P- _Q_ ___ 5086 5095 5104 513.3 5122 5131 CTG CAC GAG GCT CTG AAG ATC TGC AAT TAC ATC T7'T ACC GTC A':C TIT GTC TTr L D E A' L K -__ _~_ N _Y _I_ _E _~_ V ___ F V F
5140 5149 5158 SIE~7 5176 5185 G_yG TCA GTT TIC A~ CTT GTG G.:.C TTT GGC TTC CGC CGT T.tC TI'C CAG GAC AGG
E S V F K- L V A- -F- G F R R F- F rQ -D R
5394 5203 5212 .5221 5230 5239 2GG AAC CP.G CTG G.=~C CTG GCT ATT GTG C'FT CTG TC:C ATC ATG GGC ATC ACA CTG
W N Q ~ -D' 'L" _A ___ _ _ _~ _~ _S_ _I_ _M _ E E I E V N A S L -p- -T -N
5302 5311 ~. 5320 . 5329 533 8 5347 ATG AGG GTG CTC CGC ATT GCT CGA GIT CTG AAG CTG TtG AAG ATG GCT GTG GGC
M R V L R I A -R V L- K L- -L K M A V G

M R A L -L -D- -T V M Q A L p Q '~

GGA CTT CTC TTC ATG TrA TFG TI'T TTC ATC TIT GCA GCT CTG GGC G'IG GAG CTC
G L L F M -L Lr _~._ _F_ ___ -F A' A L G V E L

TTT GGA G.AC CTG GAG TGT.GAT GAG ACA CAC CCT TG'r GAG GGC TIG G~~T CGG CAT
FGDLE-CDE~T-HPC~E-GLGRH
5518 552? 5536 554'5 5554 ~ 5563 GCC 'ACC TTT AGG AAC TIT GGT ATG GCC TIT CTG ACC CTC ~j~,~ ~ ,(~ r~C ACT
A T F R N F G M A F L -T -L -F -R V- S T

WO 00/15845 _ g _ PCTIUS99I19675 DNASIS DPL1 Traaslatioa IT-INS]

GGT GAC AAC 'LAG A'AT G,"T ATT ATG AAG GAC ACC CTC CGG GAC 1GT GAC C~.G. GAG
G D N W ~ 'G' -__ _ _K _p.. T L _R _ _C- ~D Q E
5626 5635 5644 '.9653 5662 5671 TCC ACC TGC TAC AAC ACT GTC ATC ZCC CCT ATC TAC TTT GTG TCC TTC GTG CTG
S T C Y N T V I yS P I Y F V S F V ~L

ACG GCC CAG TTT AaC GTG ATA GCT CT~v AAG CAC
GTG CTG GTC GTC GTG AT~v C'IC'., T A Q F V L V' N V V I A V L M K H L

5734 ~ 5743 5752 5761 57?0 5779 GSA GAA AGC AAC AAG CAG GCC C.AGGAG GAG C2G
AAA GAG GCC GAG CTC GCC GAG

E S N' K E A Kr E E A E L~ E A E L E

5788 5797 5806 !i8lS 5824 ~ 5833 CTG GAG ATG AAG CCG CAG CAC TCC CTG AGC CCC
ACG CTC AGC CCC CCG GGC TTC

L E M K T L S P Q P H S P L G S P F

5842 5851 5860 ~ 5869 5878 588?

CTC TGG CCC GGG GTC AAC CCT GAC CCT CCT GGG
GTG GAG GGT AGT AGC AAG GCT

L W P G V E G V !N S P D S P K P G A

5896 5905 5914 :1923 5932 5941 CCA CAC ACC ACT GGA GCA TCG GGC TCC GAG CAC
GCC CAC ATT GCC TTC CTT CCC

P H T T A H I G A A S~ G S L E H P
F

5950 5959 5968. :1977 5986 5995 ACG ATG GTA CCC GAG GTG GTC CCC Cv.A GAC CTG
CAC CCC GAG CCA CTA CCc'l~C2G

T M V P H P~ E ~E ~ V V P L G P wD L
P ' L

6004 6013 6022 ti031 6040 6049 ACT GTG AGG AAG AGC CGG CrIC.TGTCCC GAC AGC
TCT GGT GTC ACG CTG A~.T TAC

T V R K S G aV S~ Rr H S L P N D Sr ~T Y

6058 6067 6076 ti085 6094 6103 ATG TG: CGC AAT GCT GAG TCC CTA CAC.AGGGGC TGG
GGG AGC ACT AGA GGA GGG

M C R N G $ !T _Ay E S L 'G ~H G W rG
R R

6112 ~ 6I21 6130 ti139 6148 6157 CTC CCC AAA GCC TCC ATC TCC GTT TCC CCA GCA
CAG TCA G~vC TTG CAC CAA GAC

L P K A Q S ~G S I L S V H ~S ~P A
'Q D

6166 6175 6184 ti193 6202 6211 ACC AGC T~ ATC CTA CCC AAA GTG C.~CCTG CAG CCT
CAG CTT GAT TAT CTC CAT
_ -L_ _Q _~ _p _K v _H, T S C I _D _ 6220 . 6229 6238 ~ ti247 6256 6265 G~ GCC CCC ACC TGG ATC CCT CTA CCC CCT CGC TCC
GGC.GCC AAA CCA GGC CCT

G A P T W Gw _ WO 00/15$45 _ lp _ PCT/US99/I9675 DNaSIS DNA Traaslahion [T-INS7 6274 6283 6292 6:301 6310 6319 C2G G:.T CAG A~ CCT CTC AGG CGC CAG GCA GCA BATA AGG ACT GAC TCC C2G GAT
L A Q R ~p L R R Q 'A A I _R -T D S
6328 6337 6346 6:355 6364 6373 GTG C.AG GGC C"TG G~vT AGC CCris GAA GAC C"PG TTG 'L'Ca CsG GTG ACoT G:.v CCC
TCC
V Q G L G S R E ~ _~ y °S_ _E _V
6382 6391 6400 6409 . 6418 6427 2GC C(=T CTG ACC CGG TCC T~ TCC TTC TC~ G.:~C GGG TCG AGE. ATC C~ GTG C=.G
C P L T R S S S F W G G S S I Q V Q

6436 6445 6454 ~ 6463 6472 6481 C.AG CGT TCC GGC ATC C.'AG AGC AAA GTC TCC G.:.C
A?G t.'..~1C ATC CGC CTG CCA CCT

Q R S G 'I Q -S

6490 6499 6508 6;117 6526 6535 TGC CC.y GGs. CTG C..AA CCC AGC TGG GCC AAG AGC
(;~C CCT CCA GAG ACC AGA AGC

C P G L E

6544 6553 6562 6..i71 6580 6589 TTA Cz~G CTG (iAC ACG GAG CTG AGC ZGG ATT AGC
TCA tzGA C,AC CTC CTT CCC AGC
' D T E ~L' ~~ _w -I - - ..G _D _ _ _ _ _ L E L _ 6598 6607 6616 ~ 6fi25 6634 . 6643 CAG GAA G.~.i1 CCC CTG TCC CCA CGG GAC CTG GAG
AAG AAG TGC TAC P,GT GTA ACC

Q E E P _L _S _p_ _~

6652 6661 6670 6Ei79 60'88 6697 CAG AGC 2GC AGG CGC AGG CCT GGG TvC TGG CTA CAC
CiP.T GAA CAG CGG AGA TCC

Q S C R '~ R' _p_ _ 6706 6715 6724 ~ 6733 6742 6751 ATT GCT (iTC AGC ZGT CFG GAC AGC GGC TCC CAA AGC
C;CC CGC CTA TGT CC:A CCC
~

I A V S -P
S C L D S G! 5 'Q .P- R L C P

6760 6769 6778 6i'87 6796 6805 GG~v AGC CGG CCT A'rG AAA
' S S L G -Ky G- Q ~P~. L ~G G P ,G S R P wK K

68'14 6823 6832 6E141 6850 6859 CTC AGC CCA CCC AGT ATC TCT ATA GAC CCC CCG CGG
(:~G AG:. CAG GGC TCF CCC

L S P P S

CC:A TGC AGT CCT Gv'"F GTC TGC CTC AGG AGG TCT
AGG (:iCG CCG CsCC AGT GAC AAG

P C S P G~ V C L 'R R R .A P A S D S ~K

GAT CCC TCG GTC T'C AGC CCC CTT GAC AGC ACG CC.A
(:.CT C,CC TCA CCC TCC AAG
r r D P S P-V S K
S ~P L D 'S T "A YA' S~ P 'S

WO 00/15845 _ 1 ] _ PCTIUS99/19675 DNASIS DNA Traaslatioa (T-INS]
6976 6985 6994 7003 ?012 7021 AaA GAC GTG AGT CTC TCT G~~T TTG TCT TCT A2G GAC CCC
ACG G~?~C CCA ACA GAC

K D T L ST L S G L S S LI P T A M D P

7030 7039 7048 705.7 7066 7075 TGA GTC CCC ACT CTC CCC CST G~.C CTT TCT GAT CCT AG;.
CTA CC:A CCG GGT GCA

'* V L P T~ L rP H H L S E' P G A D P S

?084 7093 7102 71L1 7120 7129 TCC GCC TGG GCA GCG.TTT CTG A.aA AGT CCC AGC AGC G3C
TCC AC:G TAA GCA GCA

S A S W A A F~ L ~K S P ~' *' A A S S H

G?G GG~1 CAC CTG CCT TCT TCA GIG GCT GGT GAG AAC TTC
CCT GG~G C.~T G.~C CAG

E A p H L p S S V A G G. D D E Q N F

?192 7201 7210 72L9 7228 7237 CGG AGA GAT C'TG A.aG AGA ACA GAG CCC 2GG CGG GSA G1.~
GTC AGC CCC ZG.:. CTC

R R V D L K R T' Q P W S. P C L R E E

~ r 3 5~

G.xA AAA GAA AGC CCA GTG TGG CG~ AGG CTC Gv.~T G /3 ' GGA CC:G ACA CCA GGa _W _P _R _~ _E _ _ _ _G_ _A~~ ~ 56u~
x ~ 4 WO bb/15845 _ 12 _ PCT/US99/~9675 ~~.3 5E8 ~9 ,] 10 20 . 30 40 50 60 GC2GAGC TGdACTGGCC CTCCTGGGGA C7'CAG:AAGCTCTCTAGAGC CCCCCACATG

70 80 90 100 lla 120 CTCCCCCACC GGGGTCCCCC GG2'IC~CG1~1TCTG~GGGGC TCCG..~TCGCC
GGr.CACCTCC

$i4lf of CCTCf'TC GACG.~.GG
GGA CCCCCCGGGG CCCCGGCTGG CCAG? AC~:A~GGAGC Gp iA' IG

190 200 210 22a 230 24a G:~GCGCCGaG G,a.GTCGGG~.C AGCCCCGTAG CT~CGCAG CTCAACGACC TGTCCGGGGC

CGGGGG:.CGG CAGGGGCCGG GGTCGACGGA A~AG:aACCCG GGC~GCGCGG ACTCCGAGGC

GGAGGGGCTG CCGTACCCGG CGCTAGCCCC GG2GGT'rTT~ TTCTAC2'IC'xr1 GCCr'1GGACAG
370 380 390 400 410. 420 CCGCCCGCGG P.GCTGGTGTC TCCGCACGGT CTGTAACCCG TG~"TTCGAGC GAGTCAGTAT

G.~IGv~TCATT CTTCTCAACT GTGTGACTCT GGV~TsIT"TTC AGGCCG'i'GTG AGG.~CATTGC
490 5a0 510 520 530 540 CIG1GACTCC CAGCGCTGCC GGATCCTGCA GGCCTTCG?~T GACI'TCATCT TFGCCTTCTT
550 560 5?0 580 590 6a0 TGCTGTGGaA A'IGGZGGTGA AGATGGTGGC CT'1GGGCATC T2'PGGGeIAGA'A~TGTTACCT

GGGAGACACT TGG~.'1ACCGGC TTGACTTTTT CATTGTCATT GCAGGGATGC TGGAGTATTC
67a sao s9o 700 710 720 GCTGGACCTG C.~iGAACGTCA GCTTCTCCGC AGTCAGGACA GTCCGTGTGC TGCGACCGCT
73a 740 750 760 ?70 780 CAGGGn~.CATT RACCGGGTGC CCAGCATGCG CATTCTCGTC ACATTACTGC 2GV:~'1C?.CCTT
790 ~ 800 810 820 830 840 GCCTATGCTG GGCAACGTCC TG~ TTTCTI~GTC TTTTTCATCT TTGGCATCGT
850 860 ' 870 880 890 900 GGGCGTCC~G CTGTGGGCAG GACTGCTICG CAF1CCGATGC TTCCTCCCCG AGAACTTCAG

CCTCCCCCTG AGCGTGGACC TGGAGCCTTrI TTACCAGAG~ GAGAA2GAGG ACCACrAGCCC
970 980 99a 1000 1010 1020 CT'ICATCTGC TCTCAGCCTC GGGeIGeIA'TGG CATGAGATCC TGCAGGAGTG 2GCCCACACT
1030 lo4a loss loso la7o loss G=GTGGGV~iAA GGCGGTGGTG GCCCACCC2G CAG2CTGGAC TATGAGACCT A'LAACAGTTC
1090 1100 1110 1120 1130 ~ 1140 CAGCAACACC ACCTGTGTCA ACTGGAACCA GTACTATACC RAC2'GCTCTG CGGGCGAGCA

CAACCCCTTC AAAGGCGCCA TCAACTTIGA CAACATTGGC TATGCCTGGA TCGCCATCIT

CCAGGTCATC ACACTGGAGG GCTGGGTCG~. CATCATGTAC TTCGTAATGG ACGCTCACTC

WO 00/15845 - l~i - PCT/US99119675 DNASIS
T-INS

CTTCTACAAC TTC.aTCTACT TCATTCTT'L'r TC-aTivATCAA
Cr':TCrZTCGTG ~GsCTVCTICT

CCTGTGCCTG GTGGTGAfiTG CCACGCeIGTr CTCCGaCACCAGaGT'CAGv.~T
.AnACAG.~GGG

1390 1400 1410 _x.420 1430 1440 GATGCGCov~AG CAGCGTGTAC GATTCCTGTC C3ATG.."TAGC.Gv.'TTCTCI'GA
.rICCCTC~ .-cCcaGGcACC TGcTATGACG AG..wTACTCaa GTACCTCCTCGa~aaAGC~cc 'rACATCCTCC

CCGAAGGv."TG t.;CCCAGGTCT CTAGGCn."TAT WGCAGCCC
AG:a:.GTCiC~ C-v."TC'GC

AGTv~C-CCCGT AGTGuGCFIGG AGCCCCAGCC GaC.aCCGTCG
C?GTu~"~GC 'rGC.aCTCGCT

TCTGTCTGTC CACCACCTGG TCCACCACCA TCACCaCCACACCrICCTG~vG
t:.aTCACCACT

1690 1700 1710 1720 1?30 1740 TAAZGGGACG CTCAGaGTTC CCCGGGCC?.G CCCaGAGATCATGCCAATGG
t:AGGACAGGG

GTCTCGCCGG CTC.aTuCTAC CACCACCCiG T.~.CACCCACT CCCT~TGGGG GCCCTCCts2G
181.0 1820 1830 1840 1850 1860 GCvTGCGGAG TCTGTACACA GCTTCTACCA TGCTCACTC-C t:At:TTGGAC',C CaGTCCGTTG
1870 . I880 1890 1900 1910 1920 CCAGGCACCC CCTCCCAGAT G:.CCATCGGA GvCAT~TGGT A6GACT~FGC, G2~GTGGGr~A

GGTGTACCCC ACTGTuCATA CCAGCCCTCC ACCAGAGATA CTGaAGG.aTA RAGCACTAGT

~?GGTGGCC CCCAGCCC'tG t;,GCCCCCCAC CCTCACCAGC '.LTC?,.aCATCC C~.CCTGGGCC
2050 2060 20?0 2080 2090 2100 CTTCAGCTCC ATu~CACAAGC TCCTGGAGAC AG~GaGTACG t;GAGCCTGCC ATAGCTCCTG

CAAAATCTCC AGCCCTIG.."T CCAAGGCAGA CAGTGGAGCC '.CGCGGGCCGG ACAGTTGTCC
21?0 2180 2190 2200 2210 2220 CFACTGTGCC CGGACAGGAG CAGGAGAGCC AGAGTCCGCT t~ACCATGTCA TGCC2GACTC
2230 2240 '2250 2260 2270 2280 AGaCAGCGAG G'~TGTGTATG AGTTCACACrI GaaCGCTCAG t.'.ACAGTGACC TCCGGGATCC

CCACAGCCGG CGGCGACAGC GGAGCCTGGG CCCAGrITuCA t'saGCCTAGiT CT'GTGCTGGC

TTTCTuC~IGG CTGATCT~TG ACACATTCCG GAAGATCGTA tsATAGCAAAT ACTTTGGCCG

GGGAATCATG ATCGCCATCC TvGTCAATAC ACTCAGCATG t'~.,CATCGAGT ACCACGAGCA
2470 2480 ~ 2490 2500 2510 2520 t,aCCCGAGGAG CTCACCAACG CCCTGGAAP.T CAGC3iACATC t~TTCAtrCA GCCTCTTCGC

2530 2540 2550 250'0 2570 2580 .
CTTC,vAGA2G CTGCTuAk.C TC~.'"'ITGTCToI CGGTCCCTTT GuCTACATTA hGnATCCCTA

CAACATCTTT Gr~TGva~TGT'CA Ti~T".o.JTCIT C_~1GTG2GIGG GAGiaTTGTGG GCC~GC~GGv 2650 20'60 2670 2680 2690 2700 AGG'I~-GCCTG TCCvi'G~."TGC GGrCCTTCCG CCTGrITV,CGG G'F~.~TC,r~IGC TCv~TGCGCTT
2710 2720 2730 2?40 2750 2760 CCTGCCvGCCCTGCAGCGCC AG~.~TCGiGG"T G."TC~~1.~GACG"'~ivG:.CAC
ACCWGGACA

CTTCTGCATGCTCCTCATGC Tv~~TCZT Cy~_'G'T~GCTC-C3TCTCTT
AT_CCTS:,GGCA

T~"TTGG~r?GTTCGC3TCTG i~.'-.CGuCa~TG.:T AG.~.1TTTCGe~.
G:~nC~CGTTG CC.~IGaCCGGeI

CTCCCTG...~TCTGGGCCATCG T~~=.CTG:CTT TCnG~sTTCLGACT'C'~G?ATrle~
ACTCitGGa.:~G

AGTCCTCTACAACGGCATGG CC~_CCACATC GTCTTGCV~CTTC.~TCG.:.CCT
G...~TCTZTACT

C~2GACTTTTGGCAACTATG T".~CTCTTTAA CCTGCT~~2GTC~AGGATF
GCCATTCTTG

CC.tIGGCAGAGC~i,.~.TCG.;CA A.=iCG:~Ga.~ GTATTCAGCT
TuCGrIGTGGa CAG'T'TRAGv."T

GCCTGTCAACTCTCAGGGGG G:-aGATGCCAC CAAGT~TGAGA'I"ITCTIZ'TC
Z'CAGAGCCTG

GCCC~GTGTGGATuGTGATG GC~CAG.iIA.~. GrIAGCGCTTGCTI1GGGAGA
GCCCTGGTGG

ACnCGCG:u~ACTACGdAe~.GA GCCTTTTGCC ACCCCTCATCC iGCGACACC
A'I~CAT.'~CGG

AATG'TCACF'ACCCAAGAG.~T CCnGCACAGG TG1'GGGGGrIACiGGCTCTCG
GCACLGGGCT

ACGTACCAGTAGCAGTGGv"T CCGCTGACri.C TGGAGCZGCCTC?AATCTCC
CACCATGeIGA

GCCAAGTGCCCGCAGCTCCC CGCACAGTCC CTGGAGTGCGGGACCAGCAG
GCAAGCAGCT

GCGCTCCAGCAGvAACAGCC TGGGCCGvGC CCCCAGCCTAGCCCGAGCG~v AAGCG~ve~GGA

GGAGCGGnGGTCCC2C...~TGT CT~.~GeIGnG:~ AC~G~uIAGTTC
CC=IGGAGAGT CAGGATGAGG

AG~GaGGACCGGGCCAGCC CAGCAGGCAG TGACC~TCGCCCTTGGAACG
CACAGGGGTT

TGAGGCCAAGAGTTCCTTTG ACCTGCC2'GA CAC,'TCTGCeIGT~CCGCAC
GTGCCGGn,C

AGCCAGCGGCCGGAGCTCTG CCTCTGAGCA CCAAGACTGTCGGCZTCAGG
AA2GGCA<1GT

WO 00/15$45 ~ _ lg _ PCTNS99/196'15 GCGTTTGGCC CGCACCCTGA GGACTGATGA CCCCCAACTG GATGGGGATG ATLs.=sC.~ATG.'~

TGAGGGAnAT CTGAGCAeIFaG GGGeIACGCAT ACAAGCCTGG GT~..AGnTCCC GGCTTCCTGC

CTGTTGCCGA GAGCGAGATT CCTGGTCGGC CTATATCTTT CCTCCTCAGT CrlAG"~TTI'CG

TCTCCT~'TGT CACCGGATCA TC~1CCCACAA GATGTTTGAC C.AiGTGV"TCC TCGTCATCAT

CTTCCTCaAC 'm,TATCACCA TCC,v.~TATG~1 GCG.:CCC.~,e~A ATiGACCCCC
ACAG.:.G..."TG~1 GCGCATCTTC CTGACCCTCT CCAACTACAT GTTCACGGCA GTCTTTCTAG C1G~.AA.TGaC

AGTGAAGGTG GTGC-uICTGG G~.~?~.,GTGG"IT TGGGG~aGCAG GCCTACCTGC GCAGCAGCTG

Gel? 2GTGCZG Ge~CGGCTPGC TGGTGCTCAT CTCCGTG'~TC G.~Ce,TCGTGG 2CTCC~I2G~a~T
4270 ~ 4280 4290 4300 9310 4320 CTCCGACAGC GGCACCAAGA T~C'~'1'G~~CAT C~n."TGAGG3TG C iGCGG~."TGC T CGGACCCT

GCGTCCACTC AGGV~TCATCA GCCGGGCCCA GGGcaCTGeI.AG CTGGTGGTAG AGACT'CTGAT

GTCATCCCTC AAACCCATTG GCA~1CATIGT GGTCATTTGC TGTGCCTTCT TC~1TCATTTT

TGGAATT"'T'C GGGGTGCAGC TCTTCAAAGG GAAGI'TCTIC GTGTv~TCAGG GTGAGvaACAC

CAGGA.a.CATC ACTAACAAAT CCGACTGCCri. TGAGGCCAGC TACCGATGv~G TCCGGCACAA

GTACrIACTTT GACAACCTGG GCC:AG~vCTCT GAT'GTCCCTG TT~GTG~,."TGG CCTCCAAGGA

T'".,GTTGuu"TT GACATCA2GT ATGAT~GC'.T GGATGCTGTG GGTGTGGATC AGCAGCCCAT

CATGA.iICCAC AACCCCTGGA TuCTGCTATA CTTCATCTCC TTCCTCC2CA TCGTGGCCTT

CTTTGTCCIG AACATGTFTG TGGGCGTGGT GG2r;GAGAAC TTCCATAAGT GCAGACAGCA

CCAGGAGGAG GAGGAGGCGA GGCGGCGTuA GGAGAAGCGA CTACGGAGGC TGGAGAAAAA

GrIGAAGGAAT CFAATGTIGG ACGATGTAAT Tv~CTTCCGGC AGCTCAGCCA GGGCTGCGTC
4930 4940 ' 4950 4960 4970 4980 AGAAGCCCAG TGCAAGCCCT ACTACTCTGA CTACTCGAGA TTCCGGCTCC TT"TCCACCA

CCTGTGTACC AGCCACTACC TGGACCTCTT CATCACTGGT GTCATCGGGC ZGAACGTGGT

WO 00/15845 _ 1~ _ PCT/US99/19675 5050 5060 5070 508() 5090 5100 CACTA'IGG~.C ATGGAACATT ACCACsCaGCC CC1G?.TCCTG GACC,dGCn."'i~ TGn,AGr'~T'CiG
5110 5120 5130 5i4t) 5150 5160 CaATTACATC TTTnCCCiTCA TCTTTG'TC2'T TGAGTCAC'T'.'C TZCAkIC~IZG Tu~GCG'I'1'n'oG
5170 5180 5190 520() 5210 5220 CTTCCGCCGT T'TCTTCCAGG ACAGGTGGa?. CCAGGZGGAC CTu:~.~TATIG

CATC~TuGGC ATCAC.tICTGG AGG:,GaT~. Cu~TCAATGCT TCCri.TG:.CC.3 TCAACCCCAC
5290 5300 5310 532() 5330 5340 C? TCATCCGT ATCATG~GGG TGCTCCGCAT 2G~"'TCGaGT'J." CTG~'~G~."~GT TC,?~.GaTGGC

iGTGGGCATG CGGGCACTGC TGGACACGGT GATuCAGGCC C2GCCCCAGG TGGGGAACCT

GGGACrTCTC TTCATGTTAT TGTTTT'TCAT CTTTGC~'><G~"7.' CiGGGCGTGG AGv.~TCTTTGG
5470 5480 5490 550(1 5510 5520 AGACCTGv~AG 2GTG.~.TGr~GA C~1C~CCCTIG 'triAGGGC2TCi C~GTCG~'ATG CCACCTTTAG
5530 5540 _°550 5S6C1 5570 5580 GAACTTZGGT ATGC-CCTTTC TGrICCCTCTT CCCY1GTCTCC: ACTGGTCir~C~ ACIG~vAATGG
5590 5600 5610 562(! 5630 5640 TATTATGA?iG CrrICACCCTCC GGGAC2CTG? CCAGGAGTCC: ACC:TGCTACA AC?.CT ,~T

CTCCCC~~LTC TACTTTGTGT CCTTCG'IGv.~T C~CGGCCCACP TTTGTG..."TGG TCAACG'E~GT
5710 5?20 5730 5740 5750 5760 CATAGCT~TG CTGATGaAGC ACC:TGGAAGA AAGCAACAAF~ GAGv~CCAAGG AGGAGGCCGA
57?0 5780 790 5800 5810 5820 C~CT~GAGGCC CxIGCTGGaGC TuGC~Gc'ITC~e~. CxACCsC'FCAGC: CCC~CAGCCCC: ACTCCCCGCT
.5830 5840 5850 5860 5870 5880 GGGCAGCCCC TTCCTCTGGC CCGGGGTuCA GGGFGTCAAC: AGiCC'1GACA GCCCTAAGCC
S890 5900 . 5910 5920 5938 5940 TvGGGCTCC:A C:ACACCACTG CCCACATTGG AGCAGCCTCG GGCT'fCTCCC TTGAGCACCC
5950 5960 5970 ~ 5980 5990 6000 CACGATGGTA CCCCACCCCG AGG? C~G'IGCC AGTCCCCCTA. GGACCAGaCC 2GC:2GAC.'TGT

GAGGAAC'TCT C~GTGTCAGCC GGACGCAC2G T"TGCCCAAT GACAGCTACA TC"Lr,CCGC~Pa TGGGACiCACT CaCTCuIGAGAT CCCTAGGAC:A CAGGGGCTGG CaGGC.'1CCCC:A AAGCCCA~"1C

AGGCT'CC:ATC TTGTCCGTTC ACTCCC.AACC AGCAGP.CACC' AGC7.GCATCC TACAGClTCC

CAAACarITGTG CACTrITCTGC TCCAGCC:TCA TGGGC,'CCCCC' ACCT aGC'~GCG CCA2CCCTAA

ACTACCCCC:A CCTGGCCC~CT CCCCT~2GGC 2CAGRGGCCT~ CTCAGGCGCC AGG~CAGCAAT

WO 00115845 _ 17 . PCT/US99/I9675 AAGGaCTGAC TCCCT~vGaTG TGCAGGGCCT G:,GTe~GCCGG GaAGACCIGT ZGTCAGAGGT

GhG2G~GGCCC TCCTGCCCTC TGACCCGG2C CTC~TCCTTC TGGGGCGGGT CGAGCATCCA

GGT~vCAGCAG CGTTCCGGCA TCC~G?GC~1 AG2'C2CCe~G CACATCCGCC TGCCAG.:.CCC

TTGCCCAGGC C7GGA~CCCA GCTGGGCCAA GGnCCCTCCrI.GAGaCCAGAA GCAG~~TTrlGa G.."~GGACACG G~1GC1'GAGC'F GGAT2TCAGG r.G;CCTCCTT CCCPGCAGCC AGG~.G~1CC

CCTGTCCCCA CGGGaCCTGA AG.~.3GTGCTAACCCAGAGCT GCAGG~.GC1~
CALGAG

G'.cTCGGTCC xcccT~TG AAcACCGGaGGcTG~rc~ccT
ac~cTCC~.Tr GTCTGGacaG

CG~v.."2~CC~ CCCCGCCTAT GTCC?.PaGCCCGGGGGCCAAC CTCTTGGGGG
CTC~rICri.CTC

TCCTGGG?G;. CGGCCTAAG~'~ AAAAACTCaGATCTCTATAG ACCCCCCGGA
CCCACCCAGT

GAGCCAGGGC TCTCGGCCCC CATGCAGTCCCTCAGGAGGA GGGCGCCGGC
TGGTGTCTGC

CAGTGACTCT AAGGATCCCT CGGTCTCCaGAGCACGGCTG CCTCACCCTC
CCCCCTTGAC

6970 6980 ' 6990 7000 ?010 7020 CCCAAAG~AA GAC~CGCTGA GTCTCTCTGGGACCCA~CaG ACATGGACCC
TTTGTCTTCT

7030 7040 7050 ' 7060 7070 7080 CTG~GTCCTA CCCACT~'fCC CCCATCACCTGGTGCAGATC CTAGCTCCGC
TTCTCCACCG

7090 7100 7110 7120 713 0 '7140 CTCCTGGGCA GCGTTTCTGA AAAGTCCCACAGCAGCCACG AGGCACCTCA
GTAaGCAGCA

7150 7160 ?1?0 7180 7190 7200 CCTGCCTTCT TCAG'I'C',GCTG GTGGGGATGATTCCGGaGAG TCGATCTGaA
CGAGCAGaAC

7210 7220 7230 . 7240 7250 7260 G~lGIr~CACiIG CCC2GGAGCC CCTGCCTCCGI~RAG~Ge~AA
GGeIAGe~IGua G=CCAGTGTG

GCCAAGGCTC CCCaCACCAG GAGCTG .......... ..........
. . ..........
.

~

~ v 3 WO 00/15845 _ lg _ PCTIUS991I9675 ~9 5?a~~ Q v9 ~;09 ( 54 C~
'ELS'TG PGDSASSLEPPTCSPTGVPRLREDTSSEGLRSPLFGPPG
APAG ~ EESG PRS~FT LNDLSGAGGR GPGSTEK
QR~DEEEDGAGA Q Q Q
DPGSADSEAEGLPYPALAPVVFFYLSQDSRPRSWCLRTVCNPWFE
RVSMLVILLNCVTLGIvtFRPCEDIACDSQRCRILQAFDDFIFAFFAV
Ehf V V KMVALGIFGKKCYLGDTW NRLDFFIVFAGMLEYSLDL QN V S
FSAVRTVRVLRPLRAINRVPSMRILVTLLLDTLPbiLGNYLLLCFF V
FFIFGIVGVQLWAGLLRNRGFLPENFSLPLSVDLEPYYQTE~NEDES
PFICSQPRENGMRSCRSVPTLRGEt:rGGGPPCSLDYETYNS S S NTT
CVNWNQYYTNCSAGEHNPFKGAIN'FDNIGYAWIAI F Q V ITLE G W V
DIMYFVMDAHSFYNFIYFILLIIVGSFFMINLCLVVIATQFSETKQR
ESQLMREQRVRFLSNASTLASFSE:PGSCYEELLKYLVYILRKAAR
RLAQVSRAIGVRAGLLSSPVARSGQEPQPSGSCTRSHRRLS VHHL
VHHHHHHHHHYHLGNGTLRVPRASPEIQDRDANGSRRLbILPPPST
PTPSGGPPRGAESVHSFYHADCHLEPVRCQAPPPRCPSEASGRTV
GSGKVYPTVHTSPPPEILKDKALV:EVAPSPGPPTLTSFNTPPGPFS S
MHKLLETQSTGACHSSCKISSPCSI~ADSGACGPDSCPYCARTGAG
EPESADHVMPDSDSEAVYEFTQDA.QHSDLRDPHSRRRQRSLGPDA
EPSSVLAFWRLICDTFRKIVDSKYFGRGIMIAILVNTLSMGIEYHEQ
PEELTNALEISNIVFTSLFALEMLLIKLLVYGPFGYIKNPYNiF D G VI
VVISVWEiVGQQGGGLSVLRTFRLNviRVLKLVRFLPALQRQLYVLM
KTMDNVATFCMLLMLFIFIFSILGDZHLFGCKFASERDGDTLPDRK
NFDSLLWAIVTVFQILTQEDWNKV.LYNGbiASTSSWAALYFIALMT
FGNYVLFNLLVAFLVEGFQAEEIGR:REDASGQLSCIQLPVNS QGGD
ATKSESEPDFFSPSVDGDGDRKKR:LALVALGEHAELRKSLLPPLII
HTAATPMSLPKSSSTGVGEALGSGSRRTSSSGSAEPGAAHHEMKS
PPSARSSPHSPWSAASSWTSRRSSRNSLGRAPSLKRRSPS GERRS
LLSGEGQESQDEEESSEEDRASPAGSDHRHRGSLEREAKSSFDLPD
TLQVPGLHRTASGRSSASEHQDCNGKSASGRLARTLRTDDPQLDG
DDDNDEGNLSKGERIQAWVRSRLF'ACCRERDSWSAYIFPPQSRFR
LLCHRIITHKMFDHVVLVIIFLNCI'CiAMERPKIDPHSAERIFLTLS N
YIFTAVFLAEMTVKVVALGWCFGEQAYLRSSWNVLDGLLVLIS VI

MSSLKPIGNIVVICCAFFIIFGILGVQLFKGKFFVCQGEDTRNITNK
SDCAEASYRWVRHKYNFDNLGQA',LbiSLFVLASKDGWVDIbIYDGL
DAVGVDQQPIMNHNPWMLLYFISFLLIVAFFVLNMFVGVY V ENF H
KCRQHQEEEEARRREEKRLRRLEKKRRNLMLDDVIASGSSASAAS
EAQCKPYYSDYSRFRLLVHHLCTSHYLDLFITGVIGLNVVTMAME
HYQQPQILD~ALKICNYIFTVIFVFESVFKLYAFGFRRFFQDR WNQ
LDLAIVLLSIMGITLEEIEVNASLPiNPTIIRIMRVLRIARVLKLLKM
A VGMRALLDTV MQALPQVGNLGLLFMLLFFIFAALGVELFG D L E C
DETHPCEGLGRHATFRNFGMAFLTLFR VSTGDNWNGIMKDTLRD C
DQESTCYNTVISPIYFVSFVLTAQFVLVNVVIAVLMKHLEES NKEA
KEEAELEAELELEMKTLSPQPHSP1:.GSPFLWPGVEGVNSPDSPKP G
APHTTAHIGAASGFSLEHPTbtVPH:PEEVPVPLGPDLLTVRKSGVS R
THSLPNDSYMCRNGSTAERSLGHRGWGLPKAQSGSILSVHS QP AD
TSCILQLPKDVHYLLQPHGAPTWGAIPKLPPPGRSPLAQRPLRRQA
AIRTDSLDVQGLGSREDLLSEVS G:PSCPLTRS SSFWGGSSIQ V Q QR
SGIQSKVSKHIRLPAPCPGLEPSWp.KDPPETRSSLELDTELS WIS G
DLLPSSQEEPLSPRDLKKCYSVETQSCRRRPGSWLDEQRRHSIAV
SCLDSGSQPRLCPSPSSLGGQPLGGPGSRPKKKLSPPSISIDPPES Q
GSRPPCSPGYCLRRRAPASDSKDPSVSSPLDSTAASPSPKKDTLSL
SGLSSDPTDMDP'VLPTLPHHLSPF'GADPSSASWAAFLKSPT'AAS
SH2071 EAPHLPSSVAGGDDEQNFRF~VDLKRTQPWSP C LR EEG KGE
SPV1VPRL'PTPG ~~
SEQ ?'t ~I

in.

WO 00!15$45 _ 19 _ PCTIUS99/t9~75 SEQ ID N0:5: SKEKQMA
SEQ ID N0:6: 5'TNGC(A/C/T}ATGGAG(C/A.)GNCC(C/T)-3' SEQ ID NO: 7 : 5' -CTT (C/G/T) CCCTTGAA(G/C)A (G/A) CTG) -3' SEQ ID N0:8: 5''-CCGCTGTCGGAGACCATGGA.GACC-3' SEQ ID N0:9: 5'-AGCGGCCCAAAATTGACCCCCACAG-3' -SEQ ID N0:10: 5'-GAAGATGCGAGTGGACAG-3' SEQ ID NO:11: 5'- CTGTGGCGATGGTCACTG-3'

Claims (60)

What Is Claimed Is:

1. An isolated nucleic acid molecule encoding a pancreatic T-type calcium channel.

2. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid is deoxyribonucleic acid.

3. The isolated nucleic acid molecule of claim 2 wherein said deoxyribonucleic acid is cDNA.

4. The isolated nucleic acid molecule of claim 3 wherein said nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:1.

5. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid molecule encodes an amino acid sequence as shown in SEQ ID NO:2.

6. The isolated nucleic acid molecule of claim 1 wherein said nucleic acid is ribonucleic acid.

7. The isolated nucleic acid molecule of claim 6 wherein said ribonucleic acid is mRNA.

8. An antisense nucleic acid molecule complementary to at least a portion of the mRNA. of claim 7.

9. A cell comprising the antisense nucleic acid molecule of claim 8.

10. An expression vector comprising the antisense nucleic acid molecule of claim 8.

11. The expression vector of claim 10 wherein the expression vector is selected from the group consisting of a plasmid and a virus.

12. A cell comprising the expression vector of claim 10.

13. A method of decreasing expression of a pancreatic T-type calcium channel in a host cell, said method comprising introducing the antisense nucleic acid molecule of claim 8 into the cell, wherein said antisense nucleic acid molecule blocks translation of said mRNA so as to decrease expression of said pancreatic T-type calcium channel in said host cell.

14. A ribozyme having a recognition sequence complementary to a portion of the mRNA of claim 7.

15. A cell comprising the ribozyme of claim 14.

16. An expression vector comprising the ribozyme of claim 14.

17. The expression vector of claim 16 wherein the expression vector is selected from the group consisting of a plasmid and a virus.

18. A cell comprising the Expression vector of claim 16.

19. A method of decreasing expression of a pancreatic T-type calcium channel in a host cell, said method comprising introducing the ribozyme of claim 14 into the cell, wherein expression of said ribozyme in said cell results in decreased expression of said pancreatic T-type calcium channel in said cell.

20. A cell comprising the nucleic acid molecule of claim 1.

21. An expression vector comprising the nucleic acid molecule of claim 1.

22. The expression vector of claim 21 wherein said expression vector is selected from the group consisting of a plasmid and a virus.

23. A cell comprising the expression vector of claim 21.

24. A method of increasing expression of pancreatic T-type calcium channel in a host cell, said method comprising:
introducing the nucleic acid molecule of claim 1 into the cell; and allowing said cell to express said nucleic acid molecule resulting in the production of pancreatic T-type calcium channel in said cell.

25. A method of screening a substance for the ability of the substance to modify T-type calcium channel function, said method comprising:
introducing the nucleic acid molecule of claim 1 into a host cell;
expressing said pancreatic T-type calcium channel encoded by said nucleic acid molecule in the host cell;
exposing the cell to a substance; and evaluating the exposed cell to determine if the substance modifies the function of the T-type calcium channel.

26. The method of claim 25 wherein said evaluation-comprises monitoring the expression of T-type calcium channel.

27. A method of obtaining DNA encoding a pancreatic T-type calcium channel, said method comprising:
selecting a DNA molecule encoding a pancreatic T-type calcium channel, said DNA molecule having a nucleotide sequence as shown in SEQ ID NO:1;
designing an oligonucleotide probe for a pancreatic T-type calcium channel based on SEQ ID NO:1;
probing a genomic or cDNA library of an organism with the oligonucleotide probe; and obtaining clones from said library that are recognized by said oligonucleotide probe, so as to obtain DNA encoding a pancreatic T-type calcium channel.

28. A method of obtaining DNA encoding a pancreatic T-type calcium channel, said method comprising:
selecting a DNA molecule encoding a pancreatic T-type calcium channel, said DNA molecule having a nucleotide sequence as shown in SEQ ID NO:1;
designing degenerate oligonucleotide primers based on SEQ ID NO:1; and utilizing said oligonucleotide primers in a polymerase chain reaction on a DNA sample to identify homologous DNA encoding a pancreatic T-type calcium channel in said sample.

29. An isolated nucleic acid molecule encoding a pancreatic T-type calcium channel, said nucleic acid molecule encoding a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence, said second amino acid sequence as shown in SEQ-ID NO:2.

30. A DNA oligomer capable of hybridizing to the nucleic acid molecule of claim 1.

31. A method of detecting presence of a pancreatic T-type calcium channel in a sample, said method comprising:
contacting a sample with the DNA oligomer of claim 30, wherein said DNA oligomer hybridizes to any of said pancreatic T-type calcium channel present in said sample, forming a complex therewith; and detecting said complex, thereby detecting presence of a pancreatic T-type calcium channel in said sample.

32. The method of claim 31 wherein said DNA
oligomer is labeled with a detectable marker.

33. An isolated pancreatic T-type calcium channel protein.

34. The pancreatic T-type calcium channel protein of claim 33 wherein said pancreatic T-type calcium channel protein is encoded by a nucleotide sequence as shown in SEQ ID NO:1.

35. The pancreatic T-type calcium channel protein of claim 33 wherein said pancreatic T-type calcium channel protein is encoded by an amino acid sequence as shown in SEQ ID NO:2.

36. An isolated pancreatic T-type calcium channel protein encoded by a first amino acid sequence having at -least 90% amino acid identity to a second amino acid sequence, said second amino acid sequence as shown in SEO
ID NO:2.

37. An antibody or fragment thereof specific for the pancreatic T-type calcium channel protein of claim 36.

38. The antibody of claim 37 wherein said antibody comprises a monoclonal antibody.

39. The antibody of claim 37 wherein said antibody comprises a polyclonal antibody.

40. A composition comprising the pancreatic T-type calcium channel protein of claim 36 and a compatible carrier.

41. A method of detecting presence of a pancreatic T-type calcium channel protein in a sample, said method comprising:
contacting a sample with the antibody or fragment thereof of claim 37, wherein said. antibody or fragment thereof binds to any of said pancreatic T-type calcium channel protein present in said sample, forming a complex therewith; and detecting said complex, thereby detecting presence of a pancreatic T-type calcium channel protein in said sample.

42. The method of claim 41 wherein said antibody or fragment thereof is labeled with a detectable marker.

43. A method of modifying insulin secretion by pancreatic beta cells, the method comprising modifying levels of functional T type calcium channels in the pancreatic beta cells.

44. The method of claim 43 wherein modifying levels of functional T type calcium channels comprises modifying T type calcium channel gene expression in the pancreatic beta cells.

45. The method of claim 44 wherein modifying T type calcium channel gene expression comprises exposing the pancreatic beta cells to a compound which modifies T type calcium channel gene expression.

46. The method of claim 45 wherein the compound is an antisense oligonucleotide targeted to the T type calcium channel gene.

47. The method of claim 43 wherein modifying levels of functional T type calcium channel comprises exposing the pancreatic beta cells to an inhibitor of the functional T type calcium channel.

48. The method of claim 43 wherein modifying levels of functional T type calcium channel comprises exposing the pancreatic beta cells to a compound which interferes with membrane T type calcium channel formation.

49. The method of claim 43 wherein the pancreatic beta cells are present in a subject having type II
diabetes.

50. A method of treating type II diabetes in a subject, the method comprising administering to the subject an amount of a compound effective to modify levels of functional T type calcium channel in the pancreatic beta cells of the subject.

51. The method of claim 50 wherein the compound modifies levels of functional T type calcium channel by modifying T type calcium channel gene expression.

52. The method of claim 51 wherein modifying T type calcium channel gene expression comprises exposing the pancreatic beta cells to a compound which modifies T type calcium channel gene expression.

53. The method of claim 52 wherein the compound is an antisense oligonucleotide targeted to the T type calcium channel gene.

54. The method of claim 50 wherein the compound is an inhibitor of the functional T type calcium channel.

55. The method of claim 50 wherein the compound interferes with membrane T type calcium channel formation.

56. A method of modifying basal calcium levels in cells, the method comprising modifying levels of functional T type calcium channels in the cells.

57. A method of modifying the action potential of L
type calcium channels in cells, the method comprising modifying levels of functional T type calcium channels in the cells.

58. A method of modifying pancreatic beta cell death, the method comprising modifying levels of functional T type calcium channels in the pancreatic beta cells.

59. A method of modifying pancreatic beta cell proliferation, the method comprising modifying levels of functional T type calcium channels in the pancreatic beta cells.

60. A method of modifying calcium influx through L
type calcium channels in cells, the method comprising modifying levels of functional T type calcium channels in the cells.