JP2002525077A - T-type calcium channel - Google Patents

Abstract

  (57) [Summary] The present invention relates to isolated nucleic acid molecules encoding pancreatic T-type calcium channels, as well as vectors and host cells containing the same. The invention further relates to methods and compositions comprising an antisense that alters expression of pancreatic T-type calcium channels. Provided is an isolated pancreatic T-type calcium channel protein and an antibody against the protein. Pharmaceutical compositions and methods of treating pancreatic T-type calcium channels are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is related to US Provisional Patent Application No. 60/098, filed August 26, 1998.
No. 004 and US Provisional Patent Application No. 60/117, filed Jan. 27, 1999.
Claim 399 priority.

[0002] The subject matter of this application was made with United States Government assistance and under the National Institutes of Health Grant No. 5-20174. The United States government may have certain rights in the invention.

[0003] The present invention relates generally to calcium channel proteins, and more specifically to pancreatic T-type calcium channel proteins and uses thereof.

[0004] Various publications are referenced throughout BACKGROUND OF THE INVENTION This application, many are put in parentheses. Full citations for each of these publications are attached at the end of the detailed description. The disclosure of each of these publications is incorporated herein by reference in its entirety.

[0005] Insulin secretion from pancreatic beta cells is a major physiological mechanism of blood glucose regulation. Elevated blood glucose levels stimulate the release of insulin from the pancreas, which in turn promotes glucose uptake in peripheral tissues, resulting in lower blood glucose levels and restoring euglycemia. Non-insulin dependent diabetes (N
(IDDM) (type II diabetes) is associated with impaired glucose-induced insulin secretion in pancreatic β-cells (Beig and Moulin, 1982).

[0006] Ca ²⁺ channels, which are gated by potentials, mediate the rapid inward migration of Ca ²⁺ ions. This is the basis for the stimulation of insulin secretion in beta cells (
Void III, 1991). In various tissues, four types of Ca ²⁺ channels have been described (ie, L (P / Q), T, N, and E channels).
The purified L-type Ca ²⁺ channel has five subunits: α ₁ , α ₂ , β
, Γ, δ (Cutterall, 1991). The primary structure of the alpha ₁ subunit constitute the four homologous domains containing six transmembrane segments (cutter ol, 1988).

[0007] Rat and human pancreatic β cells have L-type and T-type Ca ²⁺ channels (Hilliart and Matteson, 1988; Davali et al., 1996). L activated at high potential and having large conductance and dihydropyridine sensitivity as one
The type of Ca ²⁺ channel is thought to be the main pathway for Ca ²⁺ influx into β-cells (Keahey et al., 1989). In contrast, T-type Ca ²⁺ channels are activated at low potentials and have small conductance and dihydropyridine insensitivity as one.

[0008] The physiology of T-type Ca ²⁺ channels in β-cell insulin secretion has been demonstrated (Bhattacharzy et al., 1997). These channels promote exocytosis by increasing electrical activity in these cells. L type and T
Under normal conditions, Ca ²⁺ channels act in concert to promote elevation of [Ca ²⁺ ] i during glucose-stimulated insulin secretion. T-type Ca2 ⁺ channels overexpressed in beta cells are responsible, at least in part, for an excessive response of insulin secretion to non-glucose depolarizing stimuli in GK rats and rats with NIDDM induced by neonatal injection of streptozotocin. (Kato et al., 1994; Kato et al., 1996). However, T-type calcium channels overexpressed over time eventually result in an increase in basal Ca ²⁺ due to their window current. Thus, depending on the number of channels and the membrane potential, T-type Ca ²⁺ channels in β cells have two effects.

Two isoforms of the α ₁ subunit of the L-type Ca ²⁺ channel have been identified in β cells (Seino et al., 1992; Yane et al., 1992). A T-type calcium channel in rat neurons has recently been cloned (Peretz-Raze et al., 1998).
). Other subunits of the T-type Ca ²⁺ channel have not yet been identified.

Given the evidence that T-type calcium channels are associated with type II diabetes, there is a need for further characterization of T-type calcium channels.

[0011] For Summary An object of the present invention, the present invention provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channels. The invention also provides an antisense nucleic acid molecule that is complementary to at least a portion of an mRNA encoding a pancreatic T-type calcium channel.

[0012] The isolated nucleic acid molecules of the invention can be inserted into a suitable expression vector and / or host cell. Expression of the nucleic acid molecule encoding the pancreatic T-type calcium channel results in the production of pancreatic T-type calcium channels in the host cell. Expression of the antisense nucleic acid molecule in a host cell results in reduced expression of pancreatic T-type calcium channels.

The present invention further provides a ribozyme having a recognition sequence complementary to a part of mRNA encoding a pancreatic T-type calcium channel. This ribozyme can also be introduced into cells to reduce the expression of pancreatic T-type calcium channels in the cells.

The present invention further provides a method of screening a substance for altering the function of a T-type calcium channel and a method for obtaining DNA encoding a pancreatic T-type calcium channel.

[0015] Further provided is an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel. The nucleic acid molecule encodes a first amino acid sequence having at least 90% amino acid identity to the second amino acid sequence shown in SEQ ID NO: 2.

The present invention further provides a DNA oligomer capable of hybridizing to a nucleic acid molecule encoding a pancreatic T-type calcium channel. This DNA oligomer can be used in a method for detecting the presence of a pancreatic T-type calcium channel in a sample, which is also provided by the present invention.

The present invention also provides an isolated pancreatic T-type calcium channel protein, and an antibody or antibody fragment specific for the pancreatic T-type calcium channel protein.
The antibodies and antibody fragments can be used to detect the presence of pancreatic T-type calcium channel protein in the sample. 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 the second amino acid sequence set forth in SEQ ID NO: 2.

The invention further provides a method of altering insulin secretion by pancreatic beta cells, the method comprising altering the level of a functional T-type calcium channel in pancreatic beta cells. The invention further provides a method of treating type II diabetes in a patient, the method comprising administering to the patient an amount of a compound effective to alter the level of functional T-type calcium channels in beta cells of the pancreas of the patient. A method comprising:

The present invention provides a method of altering basal calcium levels in a cell, a method of altering the action potential of L-type calcium channels in a cell, a method of altering pancreatic beta cell death, and a method of altering pancreatic beta cell proliferation. How to change and L in cells
Methods for altering the flow of calcium through a calcium channel are also provided. Each of these methods involves altering the level of a functional T-type calcium channel in the cell.

[0020] As used Detailed Description of the invention, the term "nucleic acid" refers to either DNA or RNA. "Nucleic acid sequence" or "polynucleotide sequence" refers to a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'to the 3' end. This includes both self-replicating plasmids, which are infectious polymers of DNA or RNA, and non-functional DNA or RNA.

“Isolated” nucleic acid refers to isolated nucleic acid and synthetic nucleic acid in a form that is substantially purified from the organism (ie, substantially free of other materials derived from the organism). .

A “homologous” or “complementary” nucleic acid sequence refers to a protein (channel) or a portion thereof, when the DNA sequence encoding the protein is present in a human genomic or cDNA library. A nucleic acid that selectively hybridizes to, forms a double helix, or binds to a DNA sequence of interest. DNA sequences similar or complementary to the target sequence, as long as they meet the aforementioned functional tests,
The sequence may be shorter or longer than the target sequence.

Generally, this hybridization is performed in 0.2 × SSC, 0.1% SDS.
Performed on a Southern blot protocol using a 65 ° C. wash. The term "SSC" refers to a citric acid-saline solution of 0.15 M sodium chloride and 20 mM sodium citrate. Solutions are often expressed as multiples or fractions of this concentration. For example, 6 × SSC refers to a solution containing 6 times the concentration of sodium chloride and sodium citrate, ie, 0.9 M sodium chloride and 120 mM sodium citrate. 0.2 × SSC is 0.2 times the SSC concentration, that is, 0.0
Refers to a solution of 3 M sodium chloride and 4 mM sodium citrate.

The phrase “nucleic acid molecule encoding” refers to a nucleic acid molecule that directs the expression of a particular protein or peptide. This nucleic acid sequence includes both a DNA strand sequence transcribed into RNA and an RNA sequence translated into a protein or peptide. The nucleic acid molecule includes both full length nucleic acid sequences and non-full length sequences derived from full length proteins. Furthermore, it should be understood that this sequence includes degenerate codons of the native sequence or of sequences that can be introduced to confer codon dominance in a particular host cell.

As used herein, the term “located upstream” refers to the case where the promoter is a nucleic acid (D
NA) refers to the ligation of the promoter upstream of the nucleic acid (DNA) sequence positioned to mediate the transcription of the sequence.

The term “vector” refers to an autonomously self-replicating circular DNA (plasmid)
And includes both expression and non-expression plasmids. When a recombinant microorganism or cell is described as harboring an "expression vector", this includes both extrachromosomal circular DNA and DNA integrated into the host chromosome. If the vector is carried by the host cell, it may be replicated stably by the mitotic cell as an autonomous structure or the vector may be integrated into the genome of the host.

The term “plasmid” refers to an autonomous circular DNA molecule capable of replicating in a cell,
Includes both phenotypes and non-expresses. Recombinant microorganism or cell is an "expression plasmid"
When it is described as harboring this, it includes latent viral DNA integrated into the host chromosome. When the plasmid is carried by the host cell, it is either stably replicated by the mitotic cell as an autonomous structure or the plasmid is integrated into the host 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 an endogenous DNA copy capable of expressing the peptide or protein of interest. The cells produce the peptide or protein because they have been genetically modified by the introduction of an appropriate nucleic acid sequence. This recombinant peptide or protein will not be found with the peptide or protein and other subcellular components normally associated with the cell producing the peptide or protein.

The following terms are used to describe the sequence relationship between two or more nucleic acid molecules or polynucleotides or the amino acid sequence of two or more peptides or proteins. That is, "control sequence", "comparison window", "sequence identity", "sequence homology", "percent sequence identity", "percent sequence homology", "substantial identity" and "substantial identity". Homology ". A "control sequence" is a sequence that is used as a basis for sequence comparison. The control sequence may be a subset of the longer sequence, for example as a segment of the full-length cDNA or gene sequence shown in the sequence listing, or may comprise the complete cDNA or gene sequence.

The optimal alignment of sequences for aligning the comparison windows is, for example, the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search of Pearson and Lippman (1988) or computerized means of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Doctor, Madison, Wisconsin GAP, BESTFIT, FASTA and TFA
STA).

As used in nucleic acid molecules or polynucleotides, the term “substantial identity” or “substantial sequence identity” refers to at least 90 percent of the sequence when two nucleic acid sequences are optimally aligned (see above). It means sharing identity, preferably at least 95 percent sequence identity, more preferably at least 96, 97, 98 or 99 percent sequence identity.

“Percent nucleotide (or nucleic acid) identity” or “percent nucleotide (or nucleic acid) sequence identity” refers to the nucleotide sequence of two nucleic acid molecules having approximately the specified percentage of identical nucleotides as being optimally aligned. Point to comparison. For example, "
"95% nucleotide identity" refers to a comparison of the nucleotides of two nucleic acid molecules that, when optimally aligned, have 95% nucleotide identity. Preferably, non-identical nucleotide positions are different due to degenerate nucleotide substitutions (the nucleotide substitutions do not change the amino acid encoded by a particular codon).

As further used with nucleic acid molecules or polynucleotides, the term “substantial homology” or “substantial sequence homology” refers to at least 90 percent of the two nucleic acid sequences being optimally aligned (see above). It means sharing 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 homology” refers to nucleotides that differ by identical nucleotides or non-identical but degenerate nucleotide substitutions when optimally aligned. (A nucleotide substitution does not change the amino acid encoded by a particular codon.) Refers to a comparison of the nucleotides of two nucleic acid molecules having approximately the specified percentage. For example, "95%
"Nucleotide homology to" refers to a comparison of the nucleotides of two nucleic acid molecules that, when optimally aligned, have 95% nucleotide homology.

As used for polypeptides, “substantial identity” or “substantial sequence identity”
The term refers to two peptide sequences that have at least 90 percent sequence identity, preferably at least 95 percent sequence identity, more preferably at least 96, 97, when optimally aligned, such as by the GAP or BESTFIT program using default gaps. It means sharing 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 polypeptides having approximately the specified percentage of identical amino acids as optimally aligned. For example, "95% amino acid identity" refers to a comparison of the amino acids of two polypeptides that, when optimally aligned, have 95% amino acid identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. For example, substitution of an amino acid having similar chemical properties, such as charge and polarity, is unlikely to affect the properties of the protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid.

Further, as used in polypeptides, the terms “substantial homology” or “substantial sequence homology” refer to two peptide sequences when optimally aligned, such as by GAP or the BESTFIT program using a 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 amino acid sequence homology” refers to a comparison of the amino acids of two polypeptides having approximately the specified percentage of identical or conservatively substituted amino acids that are optimally aligned. For example, "95% amino acid homology" refers to a comparison of the amino acids of two polypeptides that, when optimally aligned, have 95% amino acid homology. As used herein, homology refers to residue positions that are the same amino acid or that are different but differ only by conservative amino acid substitutions. For example, substitutions of amino acids having similar chemical properties, such as charge or polarity, are less likely to affect protein properties. Examples include glutamine for asparagine or glutamic acid for aspartic acid.

The phrase “substantially purified” or “isolated” when referring to a protein (or peptide) means a chemical composition that is essentially free of other cellular components. This can be either a dry product or an aqueous solution, but is preferably in a homogeneous state. Purity and homogeneity are generally measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein (or peptide) that is the predominant species present in the preparation has been substantially purified. Generally, substantially purified or isolated proteins (or peptides) will make up more than 80% of all macromolecular species present in the preparation. Preferably, the protein (or peptide) is purified to contain more than 90% of all macromolecular species present. More preferably, the protein (or peptide) is purified to greater than 95%, and most preferably, the protein (or peptide) is purified to essentially homogeneity. In this case, no other macromolecular species is detected by conventional techniques. A “substantially purified” or “isolated” protein (or peptide) can be isolated from an organism, synthetically, ie, chemically produced, or recombinantly produced.

A “biological sample” or “sample” as used herein refers to any sample obtained from a living or dead organism. Examples of biological samples include body fluids and tissue samples.

High stringency hybridization conditions are selected at a temperature of about 5 ° C. below the thermal melting temperature (Tm) of the particular sequence at a constant ionic strength and pH. This Tm is the temperature (under constant ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Usually, stringent conditions are that the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60 ° C. The combination of parameters is important because other factors, especially the base composition and size of the complementary strand, the presence of the organic solvent, i.e., the concentration of the salt or formamide, and the degree of base mismatching can significantly affect the stringency of hybridization. More important than any one absolute measure. High stringency is achieved, for example, by hybridization overnight in a 6 × SSC solution at about 68 ° C., washing with the 6 × SSC solution at room temperature, followed by 6 × SSC solution and then 0.1 × SSC solution.
This can be achieved by washing at about 68 ° C. in a 6 × SSX solution.

[0042] Moderately stringent hybridizations include, for example, 1) 3 × sodium chloride, sodium citrate (SSC), 50% formamide, 0.1 M in pH 7.5.
Pre-hybridization and hybridization of the filter with a solution containing Tris buffer, 5 × Denhardt's solution, 2) pre-hybridization at 37 ° C. for 4 hours, 3) using a labeled probe corresponding to 3,000,000 cpm. Hybridization at 37 ° C. for a total of 16 hours, 4) washing in a solution of 2 × SSC and 0.1% SDS, 5) 4 washes for 1 minute each at room temperature and 60
Washing four times, each at 30 ° C. for 30 minutes, and 6) drying and exposure to film.

The phrase “selectively hybridizes to” refers to a specific target DNA or target RNA sequence that hybridizes only to a total cellular DNA or total cellular RNA preparation when that sequence is present in the preparation. And a nucleic acid molecule that forms or binds a double strand. Selectively hybridizing is accomplished by allowing a nucleic acid molecule to bind to a given target such that it can be detected in a different manner than non-target sequences under moderately stringent conditions of hybridization, or more preferably, under stringent conditions of hybridization. It means to combine. A "complementary" or "target" nucleic acid sequence refers to a nucleic acid sequence that selectively hybridizes to a nucleic acid molecule. Appropriate annealing conditions will vary, for example, with the length of the nucleic acid molecules, the base composition, and the number of mismatches and their location on the molecule, and often must be determined empirically. See, eg, Sambrook et al., 1989 for a description of nucleic acid molecule (probe) design and annealing conditions.

When referring to a particular sequence listing, such references may include, in addition to the sequence substantially corresponding to its complementary sequence, slight sequencing errors, single base changes, deletions, It is understood by those skilled in the art that any such variant of the sequence will correspond to the nucleic acid sequence of the signal peptide or other peptide / protein to which the sequence listing relates, as it encompasses the described sequences, including the acceptance of substitutions and the like. It is readily understood and intended herein.

The DNA molecules of the present invention may be proteins that differ from the naturally occurring form (naturally occurring protein) in the identity or position of one or more amino acid residues (a deletion that does not include all of the bases that characterize the protein). Lost analogs, substituted analogs in which one or more specific residues replace other residues, and additional analogs in which one or more amino acid residues are added to the terminal or intermediate portion of the protein) and natural Also included are DNA molecules that encode protein analogs, fragments or derivatives of the protein that share the signaling properties of the product. These molecules are "beneficial" for expression by the selected non-mammalian host.
Additional tip and end DNA sequences to facilitate codon incorporation, provision of sites for cleavage by restriction endonuclease enzymes, and construction of readily expressed vectors
And the provision of sequences or intermediate DNA sequences.

As used herein, “peptide” refers to an amino acid sequence of 3 to 100 amino acids, such that an isolated peptide containing one amino acid sequence spans more than 100 amino acids. Not intended. The peptides (whether mimotope or antimimotope peptides) which can be identified and used according to the invention are preferably less than 50 amino acids in length, said peptides being 5 to 20 amino acids in length or 20 amino acids in length. ~ 40 amino acids are more preferred.

The peptides may include any naturally occurring amino acids or non-naturally occurring amino acids, including D-amino acids, amino acid derivatives and amino acid mimetics, as long as the desired function and activity of the peptide is maintained. . (L) -amino acid or (D) in the peptide
The choice of whether or not to include amino acids depends in part on the desired properties of the peptide. For example, the incorporation of one or more (D) -amino acids can impart increased stability to the peptide and allow the activity of the peptide to remain in the body for a longer time. The incorporation of one or more (D) -amino acids can also increase or decrease the pharmacological activity of the peptide.

The peptide may be cyclized, as cyclization may provide a peptide with properties that are superior to its linear counterpart.

As used herein, the terms “amino acid mimetic” and “mimic” refer to amino acid analogs or non-amino acid portions that have identical or similar functional properties of a given amino acid. For example, an amino acid mimetic of a hydrophobic amino acid retains hydrophobicity by containing a non-polar and generally aliphatic chemical group. As a further example, an arginine mimetic can be an analog of arginine that includes a side chain that has a positive charge at physiological pH, as is characteristic of the reactive group on the guanidine side chain of arginine.

Furthermore, modifications to the peptide backbone and its peptide bonds are also encompassed by the mimics or mimetics of amino acids. Such modifications can be made to the amino acid, its derivative, the non-amino acid portion or the peptide either before or after the amino acid, its derivative or non-amino acid portion is incorporated into the peptide. Importantly, such modifications mimic the backbone and bonds of the peptide that make up the peptide and have substantially the same spatial configuration and spacing as those typical for conventional peptide bonds and backbones. It is. One example of such a modification is the reduction of the carbonyl of the amide peptide backbone to an amine. For the reduction of amides to amines, see Van et al., JOC, 46: 257 (1981) and Rauchel et al., Tetrahe.
dron. Lett. , 21: 14061 (1980), are well known and available. Thus, an amino acid mimetic is an organic molecule that possesses a similar amino acid pharmacological group as that present in the corresponding amino acid and exhibits substantially the same spatial arrangement between the functional groups.

Substitution of amino acids with non-naturally occurring amino acids and amino acid mimetics described above can enhance the overall activity or properties of individual peptides based on modifications to the backbone or side chain functional groups. For example, such modifications to the specifically described amino acid substituents and exemplified peptides can increase the stability of the peptide to enzymatic degradation and increase biological activity. Modifications to the peptide backbone can similarly impart stability and increase activity.

Those skilled in the art can easily synthesize the peptide using the above sequences or formulations. Standard techniques for preparing synthetic peptides are well known in the art. The novel peptide is based on Merrifield's solid phase peptide synthesis (SPPS) method (J. Am. Chem. Soc.
85: 2149 (1964)) or modified SPPS, or the peptides can be synthesized using standard solution methods well known in the art (eg, Bodansky, M., Principles of Peptide Synthesis, Second Revised Edition, It can be synthesized using Springer-Belllag (1988 and 1993). Also, simultaneous complex peptide synthesis (SMPS) techniques well known in the art can be used. The peptide prepared by the Merrifield method was applied to an Applied Biosystems 431A-01 peptide synthesizer (
Using an automated peptide synthesizer such as Mountinview, CA or Gauteng, Proc. Natl. Acad. Sci. USA 82: 5131 (19).
85) can be synthesized using the manual peptide synthesis method described.

With these definitions in mind, the invention provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel. The nucleic acid molecule is deoxyribonucleic acid (DNA
) Or ribonucleic acids (including RNA, messenger RNA or mRNA), genomic or recombinant, isolated or synthesized from organisms.

The DNA molecule is a messenger RNA (mRNA
) Can be a cDNA molecule that is a DNA copy of

An example of such a pancreatic T-type calcium channel is the rat pancreatic T-type calcium channel encoded by the nucleotide sequence shown in SEQ ID NO: 1. The amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 2.

The present invention also provides an antisense nucleic acid molecule that is complementary to at least a portion of an mRNA encoding a pancreatic T-type calcium channel. The antisense nucleic acid molecule is RN
A or single-stranded DNA and can be complementary to the entire mRNA molecule encoding the channel (ie, the same nucleotide length as the entire molecule). However,
It may be desirable to work with shorter molecules. In this case, the antisense molecule may be 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 it preferably consists of about 20 to about 100 nucleotides. These antisense molecules are obtained by introducing an RNA molecule or a single-stranded DNA molecule complementary to at least a part of the mRNA of the pancreatic T-type calcium channel into a cell (ie, by introducing the antisense molecule). Can be used to reduce the level of The antisense molecule can base pair with the mRNA of the channel, thereby preventing translation of the mRNA into a protein. Thus, an antisense molecule to the channel may prevent translation of mRNA encoding the channel into a functional channel protein. This antisense encodes the T-type calcium channel only in pancreatic islet cells.
It may be desirable to place the antisense molecule downstream and under the control of the insulin promoter so as to prevent translation of the RNA (does not adversely affect brain or cardiac T-type calcium channels). It is also evident that 100% inhibition of T-type calcium channels is undesirable, as minimal basal levels of Ca ²⁺ need to be maintained by the T-type calcium channels.

More specifically, antisense molecules complementary to at least a portion of the mRNA encoding the pancreatic T-type calcium channel can be used to reduce expression of a functional channel. A cell having a first level of expression of a functional pancreatic T-type calcium channel is selected, and the antisense molecule is then introduced into the cell. The antisense molecule blocks expression of a functional pancreatic T-type calcium channel, resulting in the cells at a second level of expression of a functional pancreatic T-type calcium channel.
This second level is lower than the initial first level.

[0058] Antisense molecules can be introduced into cells by any suitable means. In one embodiment, the antisense RNA molecule is injected directly into the cytoplasm of the cell, where the RNA prevents translation. Vectors can also be used to introduce the antisense molecule into cells. Such vectors include various plasmid and viral vectors. See Han et al., 1991 and Rossi, 1995 for a general description of antisense molecules and their uses.

The present invention further provides a special category of antisense RNA molecules having a recognition sequence known as a ribozyme and complementary to a particular region of the mRNA encoding the pancreatic T-type calcium channel. Ribozymes not only form complexes with target sequences via complementary antisense sequences, but also catalyze the hydrolysis or cleavage of the template mRNA molecule. Although not intended to be limiting, the m targeted by a ribozyme
Examples of suitable regions of the RNA template include any homologous region identified by comparison with various T-type calcium channels, specifically, T-type channels of pancreatic β-cells.

[0060] Expression of a ribozyme in a cell can inhibit gene expression (such as expression of a pancreatic T-type calcium channel). More specifically, a ribozyme having a recognition sequence complementary to the mRNA region encoding pancreatic T-type calcium channel can be used to reduce the expression of pancreatic T-type calcium channel. Cells with the first level of expression of the pancreatic T-type calcium channel are selected, and the ribozyme is then introduced into the cells. The ribozyme in the cell reduces the expression of the pancreatic T-type calcium channel in the cell, since the mRNA encoding the pancreatic T-type calcium channel cannot be cleaved and translated.

[0061] Ribozymes can be introduced into cells by any suitable means. In one embodiment, the ribozyme is injected directly into the cytoplasm of the cell, where the ribozyme is m
Cleavage the RNA, thereby preventing translation. Vectors can also be used to introduce the ribozyme into cells. Such vectors include a variety of plasmid and viral vectors (note that the ribozyme-encoding DNA does not need to be "integrated" into the genome of the host cell; it is, for example, infected by the viral vector and ribozyme May be expressed in host cells with a vector that expresses For a general description of ribozymes and their use see Server et al., 1990; Krisei et al., 1991; Rossi, 1999; and Christophersen et al., 1995.

The nucleic acid molecules of the invention can be expressed in a suitable host cell using conventional methods. Any suitable host and / or vector system can be used to express the pancreatic T-type calcium channel. Xenopus oocytes are preferred for in vitro expression. The most suitable host cells for in vivo expression are pancreatic β cells.

[0063] The method of introducing the nucleic acid molecule into a host cell may involve the use of an expression vector containing the nucleic acid molecule. These expression vectors (such as plasmids and viruses; viruses include bacteriophages) can then be used to introduce the nucleic acid molecule into a suitable host cell. In order to express a pancreatic T-type calcium channel in a host cell, for example, a DNA encoding the pancreatic T-type calcium channel is prepared by:
The host cell can be injected into the nucleus, or transformed into the host cell with a suitable vector, or the mRNA encoding the pancreatic T-type calcium channel can be injected directly into the host cell.

Various methods for introducing a nucleic acid molecule into a host cell are known in the art. One method is microinjection, in which DNA is injected directly into the nucleus of a cell via a fine glass needle (or RNA is injected directly into the cytoplasm of the cell). DNA
Can be incubated with an inert carbohydrate polymer (dextran) to which positively charged chemical groups (DEAE, diethylaminoethyl) are attached. The D
NA attaches to DEAE-dextran via its negatively charged phosphate group. These large particles, including DNA, are now attached to the surface of cells, and cells are thought to take up them by a process known as endocytosis. Some of the DNA escapes the nucleus while avoiding cytoplasmic destruction of the cell.
Can be transcribed into RNA, like any other gene in the cell. In another method, the cells efficiently take up the DNA in the form of a precipitate along with calcium phosphate. In electroporation, cells are placed in a solution containing DNA and a short electrical pulse is applied that causes the membrane to open pores temporarily. DNA enters the cytoplasm directly through this hole, and DEAE
-Bypassing the endocytic vesicles through which the DNA passes by the dextran method and the calcium phosphate method. DNA can also be incorporated into artificial lipid vesicles, liposomes. They fuse with the cell membrane and deliver its contents directly to the cytoplasm. In a more direct approach, DNA is absorbed on the surface of a tungsten microprojectile and shot into cells using a shotgun-like device.

Some of these methods, namely microinjection, electroporation, and liposome fusion, have also been adapted to introduce proteins into cells. For a review, see Mannino and Guldo-Fogherite, 1988; Shigekawa and Dova, 1988; Kapetsch, 1980; and Klein et al., 1987.

[0066] Additional methods for introducing nucleic acid molecules into cells include the use of viral vectors. One such virus that is widely used for protein production is the insect virus baculovirus. See Miller (1989) for a review of baculoviruses. Various viral vectors, such as bacteriophage, vaccinia virus, adenovirus, and retrovirus, have also been used to transform mammalian cells.

As mentioned above, some of these methods of transforming cells require the use of a mediating plasmid vector. Cohen and Boyer US Patent No. 4,23
No. 7,224 describes the production of an expression system in the form of a recombinant plasmid using restriction enzyme digestion and ligation with DNA ligase. These recombinant plasmids are then introduced by transformation and replicated in single cell cultures, including prokaryotic and eukaryotic cells, grown in tissue culture. This DNA sequence is described in Sambrook et al.
It is cloned into a plasmid vector using standard cloning methods known in the art as described by 9).

A host cell into which a nucleic acid encoding a pancreatic T-type calcium channel has been introduced can be used to produce (ie, functionally express) a pancreatic T-type calcium channel. The function of this encoded pancreatic T-type calcium channel can be assayed according to methods known in the art (Vang et al., 1996).

Having identified a nucleic acid molecule encoding a pancreatic T-type calcium channel and a method of expressing the pancreatic T-type calcium channel encoded thereby, the present invention further provides agents that alter the function of T-type calcium channels (eg, , Compounds or inhibitors). The method comprises introducing a nucleic acid molecule encoding a 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 cells are then contacted with a substance, which is
Evaluate to determine whether to alter the function of type calcium channels. From this evaluation, substances that are effective in altering the function of the T-type calcium channel can be found. Such agents include, for example, calcium channel inhibitors, agonists, or antagonists (eg, mibefradil)
And mibefradil analogs, amiloride, NiCl ₂ , antisense molecules, and second messengers).

The evaluation of the cells to determine whether the substance alters the function of the T-type calcium channel can be made by any method known in the art. The evaluation can include directly monitoring expression of T-type calcium channels in the host cell. Alternatively, the assessment may be indirect and include monitoring calcium transport through the channel (such as by the method disclosed by Vang et al., 1996).

The nucleic acid molecules of the invention can be used as probes or as primers for cloning and then obtaining DNA encoding other pancreatic T-type calcium channels by either colony / plaque hybridization or the polymerase chain reaction (PCR). Can be used for design.

To identify colonies or plaques containing cloned DNA encoding the members of the pancreatic T-type calcium channel family using known methods (see Sambrook et al., 1989), see SEQ ID NO: 1. Specific probes derived therefrom can be used. One skilled in the art will appreciate that such probes can be used under highly stringent conditions (eg, 5
Hybridization at 42 ° C. with × X SSPC and 50% formamide.
(5 × SSPC wash at 50-65 ° C.) can be used to obtain sequences with regions of homology or identity greater than 90% to the probe. Sequences that have a lower percentage of homology or identity to the probe and that also encode pancreatic T-type calcium channels can be obtained by reducing the stringency of hybridization and washing (eg, reducing the temperature of hybridization and washing). Or reducing the amount of formamide used).

More specifically, in one embodiment, the method selects a DNA molecule encoding a pancreatic T-type calcium channel, wherein the DNA molecule has the nucleotide sequence shown in SEQ ID NO: 1, or a fragment thereof. And designing an oligonucleotide probe for a pancreatic T-type calcium channel based on SEQ ID NO: 1. The genomic or cDNA library of an organism is then probed with the oligonucleotide probe and the clone recognized by the oligonucleotide probe is used to obtain DNA encoding another pancreatic T-type calcium channel. Get from.

The specific primers derived from SEQ ID NO: 1 can be used in PCR using known methods to amplify DNA sequences encoding members of the pancreatic T-type calcium channel family (Innis et al., 1990; reference). One of skill in the art can use such primers under highly stringent conditions (eg, annealing at 50-60 ° C., depending on the length of the primers and specific nucleotide content used).
It recognizes that a sequence having a region that is 75% or more homologous or identical to the primer is amplified.

More specifically, in a further embodiment, the method comprises selecting a DNA molecule encoding a pancreatic T-type calcium channel having the nucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof. Designing a degenerate oligonucleotide primer based on the region of SEQ ID NO: 1, and using the primer in a polymerase chain reaction using a DNA sample to be screened for the presence of a sequence encoding a pancreatic T-type calcium channel as a template. Including the steps to use. The resulting PCR product can be isolated and sequenced to identify a DNA fragment encoding a polypeptide sequence corresponding to a target region of a pancreatic T-type calcium channel.

Various modifications of the nucleic acid and amino acid sequences disclosed herein are encompassed by the present invention. These altered sequences still encode functional pancreatic T-type calcium channels. Accordingly, the present invention further provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel, wherein the isolated nucleic acid molecule has at least 90% amino acid identity to the second amino acid sequence set forth in SEQ ID NO: 2. A nucleic acid molecule encoding an amino acid sequence is provided. In a further embodiment, the first amino acid sequence has at least 95%, 96%, 97%, 98% or 99% amino acid identity to SEQ ID NO: 2.

The present invention further provides an isolated DNA oligomer capable of hybridizing to a nucleic acid molecule encoding a pancreatic T-type calcium channel of the present invention. Such an oligomer can be used as a probe in a method for detecting the presence of pancreatic T-type calcium channels in a sample. More specifically, a sample can be contacted with the DNA oligomer, and the DNA oligomer hybridizes to and forms a complex with any pancreatic T-type calcium channels present in the sample. Then, the presence of the pancreatic T-type calcium channel in the sample can be detected by detecting the complex.

[0078] The complex can be detected using methods known in the art. Since the DNA oligomer is labeled with a detectable marker, detection of the marker after the DNA oligomer has hybridized to any pancreatic T-type calcium channel in the sample (the unhybridized DNA oligomer has been washed away) Is the detection of the complex. Detection of the complex indicates the presence of pancreatic T-type calcium channels in the sample. As would be readily apparent to one of skill in the art, the method could also be used to quantitatively assess the amount of pancreatic T-type calcium channels in a sample.

For detection, the oligomer may be labeled, for example, with a radioisotope, biotin, an element that does not transmit X-rays, or a paramagnetic ion. Radioisotopes are commonly used and are well known to those skilled in the art. Representative examples include indium-111, technetium-99m, and iodine-123. Biotin is a standard label that can detect oligomers labeled with biotin with avidin. Paramagnetic ions are also commonly used, including, for example, chelating metal ions of chromium (III), manganese (II), and iron (III). When using such a label, the labeled DNA
Oligomers 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 enzyme labels (horseradish peroxidase, alkaline phosphatase, etc.) and fluorescent labels (
FITC or rhodamine).

The invention further provides an isolated pancreatic T-type calcium channel protein. Preferably, the protein is encoded by the nucleotide sequence shown in SEQ ID NO: 1. The protein preferably has the amino acid sequence shown in SEQ ID NO: 2. Further, the present invention provides an isolated pancreatic T-type calcium channel protein encoded by a first amino acid sequence having at least 90% amino acid identity to the second amino acid sequence shown in SEQ ID NO: 2. I do. In a further embodiment, the first amino acid sequence is at least 95% relative to SEQ ID NO: 2.
, 96%, 97%, 98% or 99% amino acid identity.

[0081] The pancreatic T-type calcium channel molecule of the present invention may include a leader sequence for directing the pancreatic T-type calcium channel protein to a desired portion of a cell.

To express the channel in a host cell, a methionine residue is added to the amino terminus of the amino acid sequence of the mature pancreatic T-type calcium channel protein (ie, added to SEQ ID NO: 2) or ATG is added to the nucleotide sequence. It will be readily apparent to those skilled in the art that it may be necessary to add to the 5 'end of (i.e., to SEQ ID NO: 1). Thus, when reference is made to SEQ ID NO: 1 or SEQ ID NO: 2, it is specifically intended to include the methionine form of the mature channel.

The present invention further provides an antibody or a fragment thereof specific for the pancreatic T-type calcium channel of the present invention. Antibodies of the present invention include polyclonal and monoclonal antibodies capable of binding to the pancreatic T-type calcium channel, fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the invention can be made using one of the techniques known in the art, such as chimerization. Fragments of the antibodies of the invention include Fab
, F (ab ') ₂ , and Fc fragments.

The present invention also provides a hybridoma capable of producing the above-described antibody. Hybridomas are immortalized cell lines that can secrete specific monoclonal antibodies.

In general, methods for preparing polyclonal and monoclonal antibodies and hybridomas capable of producing the desired antibody are well known in the art (Campbell, 1
984 and Estee. See Gross et al., 1980). Any animal known to produce antibodies (mouse, rat, etc.) can be immunized with the antigenic pancreatic T-type calcium channel (or antigenic fragment thereof). Methods of immunization are well-known in the art. Such methods include subcutaneous or intraperitoneal injection of the protein. One of skill in the art will recognize that the amount of the protein used for immunization will vary depending on the animal to be immunized, the antigenicity of the protein, and the site of injection.

[0086] The protein used as an immunogen may be modified to increase the antigenicity of the protein, and may be administered with an adjuvant. Methods for increasing the antigenicity of a protein are well known in the art, and include the binding of the antigen to a heterologous protein (such as globulin or beta-galactosidase) or binding via inclusion of an adjuvant during immunization. However, the present invention is not limited to these.

For monoclonal antibodies, spleen cells were excised from the immunized animal,
It becomes a hybridoma cell producing a monoclonal antibody by fusing with a myeloma cell such as SP2 / O-Ag15 myeloma cell.

[0088] Any of several methods well known in the art can be used to identify hybridoma cells that produce antibodies with the desired properties. These include ELISA
Assay, western blot analysis, or radioimmunoassay (Lutz et al., 1988)
Screening of hybridomas.

A hybridoma secreting the desired antibody is cloned and its class and subclass determined using techniques known in the art (Campbell, 1984).

For polyclonal antibodies, antibodies, including antisera, are isolated from the immunized animal and screened for the presence of antibodies with the desired specificity using one of the techniques described above.

The invention further provides an antibody as described above in a detectable labeled form. Antibodies include radioisotopes, affinity labels (biotin, avidin, etc.), enzyme labels (horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (FIT
C or rhodamine, etc.), paramagnetic atoms and the like.
Techniques for performing such labeling are well known in the art. See, e.g., Sternberger et al., 1970; Beyer et al., 1979; Ingvar et al., 1972; and Gording, 1976.

The labeled antibodies or fragments thereof of the present invention can be used to identify cells or tissues that express pancreatic T-type calcium channels, to identify a sample containing pancreatic T-type calcium channels, or to identify pancreatic T-type calcium in a sample. It can be used for in vitro, in vivo, and in situ assays to detect the presence of type calcium channels. Thus, more specifically, the antibody or fragment thereof can be used to detect the presence of pancreatic T-type calcium channels in the sample by contacting the sample with the antibody or fragment thereof. The antibody or fragment thereof binds to and forms a complex with any pancreatic T-type calcium channel present in the sample. The complex can then be detected, thereby detecting the presence of pancreatic T-type calcium channels in the sample. As would be readily apparent to one of skill in the art, such a method would also be used to quantitatively assess the amount of pancreatic T-type calcium channels in a sample. As will be readily apparent, such antibodies can also be used to reduce the level of functional T-type calcium channels by blocking them. Thus, the antibodies can be used in the methods of the invention to alter the levels of functional T-type calcium channels in pancreatic beta cells.

Furthermore, there is provided a composition comprising the pancreatic T-type calcium channel protein and a carrier which is not affected by mixing.

In the methods of the present invention, a tissue or cell is contacted with or exposed to a composition or compound of the present invention. In the context of this invention, "contacting" a tissue or cell with a composition or compound or "exposing" the tissue or cell thereto, either in vitro or ex vivo, usually involves the composition or compound in a liquid carrier. To a cell suspension or tissue sample or administering the composition or compound to cells or tissues in an animal (including a human).

Methods of altering insulin secretion by pancreatic beta cells, treating type II diabetes, altering basal calcium levels in cells, L in cells
Methods of altering the action potential of a calcium channel, altering the death of pancreatic beta cells, altering the proliferation of beta cells of the pancreas, and altering the flow of calcium through L-type calcium channels in cells Wherein each of the methods comprises altering the level of a functional T-type calcium channel in the cell is provided for therapeutic use. Formulation of therapeutic compositions and their subsequent administration are considered to be within the skill of the art. In general, for treatment, a subject suspected of requiring such treatment is administered a composition of the present invention, usually with a pharmaceutically acceptable carrier, to the specific nature of the disease, its severity and general condition. It is administered in amounts and for periods that vary accordingly. The pharmaceutical compositions of the present invention can be administered in a number of ways depending on whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ocular, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous infusion or infusion, subcutaneous injection, intraperitoneal injection or intramuscular injection, for example, pulmonary administration by inhalation or insufflation, or intrathecal or intraventricular administration.

[0096] Formulations for topical administration may include skin sticking absorbents, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be required or desired. Coated condoms, gloves, etc. can also be helpful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or water-insoluble media, capsules, sachets or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desired.

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 that is known to promote uptake is Lipofectin (BRL, Bethesda, MD).

Dosing will depend on the severity and responsiveness of the condition being treated, along with the course of treatment, which lasts from days to months, or until treatment is effective, or until alleviation of the condition is 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. The optimal dose may vary depending on the relative potency of the particular composition, and can generally be calculated based on the IC ₅₀ or EC ₅₀ in in vitro and in vivo animal studies. For example, the molecular weight of (oligonucleotide sequence and / or chemical structure was deduced from) compounds, and, for example, given the effective dose of such that (experimentally obtained) IC _50, the dose in mg / kg of mechanical Is calculated.

The method of the present invention shows that the regulation of T-type calcium channels directly alters basal calcium levels in cells, which in turn modulates the activity of L-type calcium channels, which in turn translates into insulin secretion and It modulates cell death, which in turn is based on the finding that it treats type II diabetes. The methods of the present invention are further based on the discovery that modulation of T-type calcium channels directly affects basal and glucose-induced insulin secretion.

[0102] T-type calcium channels belong to the family of low voltage activated calcium channels. Altering (increase or decrease) the “level” of a functional T-type calcium channel alters the expression of the T-type calcium channel gene,
Refers to altering the activity of a T-type calcium channel, such as by inhibiting the function of the T-type calcium channel, and / or altering the formation of T-type calcium channels through the active membrane. As used herein, "functional" refers to the synthesis of a calcium channel in a cell to allow the calcium channel to properly insert into the cell membrane and conduct calcium ions in concert with the low voltage activation channel. And all necessary post-translational processing.

Accordingly, the present invention provides a method of altering the secretion of insulin by pancreatic beta cells, comprising altering the level of T-type calcium channels in the pancreatic beta cells.

The levels of T-type calcium channels in pancreatic beta cells can be altered in various ways at the level of genes and proteins and “functional calcium channels”. In one embodiment, the level is altered by altering T-type calcium channel gene expression of the T-type calcium channel in the cell.
This can be achieved by exposing the cells to a compound that alters the calcium channel gene expression of a T-type calcium channel. This compound is, for example, the T
Could be an antisense oligonucleotide targeting a type 2 calcium channel gene. In a similar embodiment, the compound that modulates T-type calcium channel gene expression of the T-type calcium channel could be a ribozyme.

Other methods of altering the expression of the T-type calcium channel gene include site-directed mutagenesis of the T-type calcium channel gene to prevent expression of the T-type calcium channel or various gene therapy techniques. Could be included.

The level of specific activity of a T-type calcium channel in a cell can also be altered by exposing the cell to an inhibitor of the T-type calcium channel. Such inhibitors include, for example, mibefradil, mibefradil analogues, amiloride, NiCl _2, and the second messenger can be mentioned that modulate the activity of said T-type calcium channels. Other inhibitors of the T-type calcium channel could be readily identified by screening methods, including those described above. In addition to chemical inhibitors, peptide inhibitors could be identified using screening methods (eg, phage display libraries and other peptide screening methods).

As used herein, “mibefradil analog” is meant to include compounds having the formula:

[0108]

[Outside 1]

Wherein R is hydrogen, alkyl, or a moiety having the formula C (O) R ′,
Is alkyl or aryl. In the above formula, the alkyl is a straight-chain alkyl, especially straight chain alkyl of C ₁ -C ₁₂ (e.g., methyl, ethyl, n- propyl, n- pentyl, n- hexyl, n- heptyl, n- octyl, n -Nonyl, etc.)
Branched alkyl, especially C ₁ -C ₁₂ branched alkyl (eg, isobutyl, isopentyl, neopentyl, hex-2-yl, hex-3-yl, hept-2)
- yl, hept-3-yl, etc.), and cycloalkyl, especially cycloalkyl C ₁ -C ₈ (e.g., cyclopentyl, cyclohexyl, cycloheptyl, 4-
Methylcyclohexyl). These alkyl groups can be substituted or unsubstituted. When substituted, suitable substituents include, for example, aryl groups, halogen atoms, hydroxy groups, alkoxy groups, carboxyl groups,
Includes amine groups and the like, and combinations of these substituents. Mibefradil analogs that are particularly well suited for blocking (inhibiting) the activity of T-type calcium channels but not for L-type calcium channels are those having the formula:

[0110]

[Outside 2]

And having the following formula:

[0112]

[Outside 3]

Wherein R ″ is an unsubstituted alkyl group or a substituted alkyl group that does not contain an alkoxy substituent. “Mivefradil analogs” may be present at other positions in the structure, such as on the phenyl moiety of benzimidazole , Is also meant to include compounds having the above formula substituted at the nitrogen of the benzimidazole at other positions of the tetrahydronaphthyl ring and the like. Compounds having the above formula wherein F is replaced by another substituent such as another halogen are also included in the meaning of "mibefradil analogs". Compounds having the above formula wherein the methyl or isopropyl group of the amine or both are replaced with another substituent such as another alkyl moiety are also included within the meaning of "mibefradil analogs". Moreover, "mibefradil analog" is meant to include compounds generally described and / or specifically disclosed in US Patent No. 4,808,605, incorporated herein by reference. I do. Further, "mibefradil analog" is also meant to include the pharmaceutically acceptable salts of the above-described derivatives. Illustrative pharmaceutically acceptable salts include hydrochloric, hydrobromic, nitric, sulfuric, phosphoric, citric, formic, maleic, acetic, succinic, tartaric, methanesulfonic, p-toluenesulfonic, and the like. It is a salt formed.

Mibefradil analogs are described, for example, in US Pat. Nos. 4,808,605, 5,91.
No. 0,606, 5,892,055, 5,811,557, 5,81
No. 1,556, and 5,808,088. Each of these is incorporated herein by reference.

[0115] The levels of T-type calcium channels in the cells can also be altered by exposing the cells to compounds that interfere with the formation of membrane T-type calcium channels.

[0116] The level of a functional T-type calcium channel can also be altered by the use of molecules that bind to the transcriptional regulator of the T-type calcium channel gene, such as the promoter region of the gene.

The invention further provides a method of treating type II diabetes in a patient (human or animal). The method comprises administering to the patient an amount of a compound effective to alter the level of T-type calcium channels in pancreatic beta cells of the patient. As described above, the compound may alter the expression of the calcium channel gene of a T-type calcium channel, or by inhibiting the T-type calcium channel, or by preventing the formation of a membrane T-type calcium channel. It may alter the level of T-type calcium channels.

In the context of the present invention, “modulate” or “modify” means either inhibition or stimulation. This modulation can be measured by methods routine in the art, for example by Northern blot assays for mRNA expression, Western blot assays for protein expression, or calcium channel activity assays.

The compounds and / or inhibitors used in the methods of the present invention can be any that can provide (directly or indirectly) a biologically active metabolite or a residue thereof upon administration to an animal, including a human. Or a pharmaceutically acceptable salt, ester, or salt of the ester, or any other compound / inhibitor. Thus, the present disclosure includes, for example, prodrugs and pharmaceutically acceptable salts of the compounds and / or inhibitors used in the present invention, pharmaceutically acceptable salts of the prodrugs, and other biologically acceptable salts. It extends to equivalents.

With respect to prodrugs, the compounds and / or inhibitors for use in the present invention may additionally or alternatively be prepared to be delivered in a prodrug dosage form. The term prodrug refers to a therapeutic agent that is prepared in an inactive form that is converted to an active form (ie, a drug) in the body or its cells by the action of endogenous enzymes or other chemicals and / or conditions.

With respect to pharmaceutically acceptable salts, the term pharmaceutically acceptable salt refers to the physiologically and pharmaceutically acceptable salts of the compounds and / or inhibitors used in the present invention, ie, the parent. Refers to salts that retain the desired biological activity of the compound and do not impart undesired toxic effects to it.

Drugs, such as peptide drugs that inhibit the T-type calcium channel or prevent the formation of functional T-type calcium channels, can also be identified by other methods. For example, a monoclonal antibody can be prepared that specifically hybridizes to the T-type calcium channel to prevent activity and / or channel formation. Once a monoclonal antibody that specifically hybridizes to the T-type calcium channel is identified, the monoclonal (which itself is a compound or inhibitor that can be used in the present invention) is a peptide capable of mimicking the inhibitory activity of the monoclonal antibody Can be used to identify One such method utilizes the development of an epitope library and the biopanning of a bacteriophage library. That is,
Epitope libraries have been developed in an attempt to define the binding sites for various monoclonal antibodies. Palmray and Smith have developed a bacteriophage expression vector that presents a foreign epitope on its surface (Palmley, S.F. & Smith, G.P., Gene, 73: 305-318 (1988)).
. This vector could be used to construct a large collection of bacteriophages that would contain virtually any possible sequence of short (eg, 6 amino acids) peptides. They also developed biopanning, a method for affinity purification of phage displaying foreign epitopes using specific antibodies (Palmley, S. F. & Smith, G. P., Gene, 73: 305-318). (1988), Virula, S.E., et al., Proc.Natl.Acad.Sci.USA, 8
7: 6378-6382 (1990), Scott, J. et al. Kei. & Smith, G. P. , Science, 249: 386-390 (1990); Bee. Et al., J MoI Biol, 227: 711-718 (1
992), Smith, G .; P. & Scott, Jay. Kei. , Methods
in Enzymology, 217: 228-257 (1993)).

Following the development of the epitope library, Smith et al. Next used the Palmray and Smith bacteriophage expression vector method and biopanning method to identify epitopes from all possible sequences of a given length. Suggested that it should be possible. This suggests that peptide libraries for antibodies could be identified by biopanning an epitope library, which could then be used for vaccine design, epitope mapping, gene identification, and many other applications. Inspired (Palmley, S. F. & Smith, G.P., Gene, 73: 305-318 (1988), Scott, J.K.
, Trends in Biochem Sci, 17: 241-245 (19
92)).

Investigators searching for epitope sequences using epitope libraries and biopanning have instead found peptide sequences that mimic the epitope, ie, sequences that did not match the continuous linear original sequence. Alternatively, a sequence was found that was not necessarily present in the native protein sequence. These mimetic peptides are called mimotopes. In this way, various binding sites and / or protein mimotopes were found.

The sequences of these mimotopes, by definition, do not correspond to the continuous linear native sequence or are not necessarily present in any way in naturally occurring molecules, ie, naturally occurring proteins. The sequence of the mimotope only forms peptides that functionally mimic binding sites on naturally occurring proteins.

Many of these mimotopes are short peptides. The availability of short peptides that can be easily synthesized in large quantities and mimic naturally occurring sequences (ie, binding sites) creates great potential applications.

Using this technique, mimotopes against monoclonal antibodies that recognize T-type calcium channels can be identified. The sequences of these mimotopes are short peptides that can then be used in various ways, for example, as peptide drugs that bind to and reduce the activity of T-type calcium channels. Once the sequence of the mimotope has been determined, the peptide drug can be chemically synthesized.

Materials and Methods Cell Culture-INS-1 cells were cultured in 10% FBS, 25 U / mL penicillin, 25 mg / ml.
RPMI containing mL streptomycin and 50 μM mercaptoethanol
The cells were cultured in 1640 medium in an atmosphere of 5% CO ₂ in air at 37 ° C. for 2 to 5 days before recording.

Preparation of islet cells—collagenase (4 mg / mL, Boehringer Mannheim, Indianapolis, Ind.), DNase I (10 μg / mL, Sigma, St. Louis, Mo.), CaCl ₂ (1.28 mM) and bovine serum albumin ( 1mg
/ ML, Gibco BRL, after perfusion in the pancreas with 2 mL of Hank's solution (Gibco BRL, Grand Island, NY)
The pancreas of Sprague-Dawley rats (Charles River Laboratory, Wilmington, MD) was removed. The pancreatic tissue was incubated at 37 ° C. for 20 minutes, and then washed five times with Hank's solution containing no enzyme. The islets were removed and treated with 0.1% pancreatin (Sigma) at 37 ° C. for 5 minutes. After obtaining single cells by crushing the islets with a plastic pipette tip, they were transferred to a 35 mm culture dish. Cells were cultured in RPMI 1640 medium (Gibco BRL) containing 5 mM glucose, 10% FBS and P / S for 3 hours.
The cells were cultured at 7 ° C. and 5% CO ₂ for ₂ to 5 days before the experiment.

RNA Isolation-Total RNA was isolated from cultured INS-1 cells and various freshly excised rat tissues by the guanidinium isothiocyanate / phenol method (Komzinsk and Satch, 1987). Poly-A-RNA is oligo (dT) twice in succession
Total RNA was isolated by running on a cellulose spin column (Ambion, Austin, Tex.).

CD encoding the α ₁ subunit of the T-type Ca ²⁺ channel in INS-1
NA cloning-First strand cDNA was prepared using poly dT primer and 2 μg of INS-1 cell mRN
Prepared using A and M-MLV reverse transcriptase (Gibco BRL). Degenerate primer (forward) based on the voltage-dependent Ca ²⁺ channel α ₁ subunit sequence conserved in domain III (SEQ ID NO: 6) 5′-TNGC (A / C / T
) ATGGAG (C / A) GNCC (C / T) -3 'and (reverse) (SEQ ID NO: 7) 5'-CTT (C / G / T) CCCTTGAA (G / C) A (G / A) C
The first 433 bp DNA fragment of the channel was estimated by PCR using TG-3 '. Marathon (TM) cDNA Amplification Kit (Clontech, Palo Alto, CA) using a rapid amplification -PCR (RAC of 3'- and 5'-cDNA ends in order to obtain the entire gene of the channel alpha ₁ subunit
E-PCR) was performed. For 5'-RACE-PCR, the forward primer is an adapter primer and the reverse primer is (SEQ ID NO: 8) 5 '
-CCGCTGTCGGAGACCATGGAGACC-3 '. 3'-
For RACE, the forward primer was (SEQ ID NO: 9) 5'-AGCGG
CCCAAAATTGACCCCCCACAG-3 'and the reverse primer was poly dT. The RT-PCR product was subcloned into a pT-Adv vector (Clontech), and a dideoxynucleotide sequencing assay was performed using a dsDNA cycle sequencing system (Gibco BRL).

Tissue Distribution—RT-PCR assays were used to examine gene expression of T-type Ca ²⁺ channels predicted from β cells in rat brain, heart, kidney, and liver. The primer used for this RT-PCR was (SEQ ID NO: 10) 5′-GAAGATGCCGAGTGGAC.
AG-3 '(forward) and (SEQ ID NO: 11) 5'-CTGTGGCGATGG
TCACTG-3 '(reverse direction). The PCR product was detected on a 1% gel by agarose gel electrophoresis.

Genome walking-In a primer nested PCR reaction using the gene specific primer (GSP) and the adapter primer (AP) attached to the kit,
A genome walker library (Clontech) was used as a template. 1st P
The CR reaction was performed in five tubes. Each tube has a total volume of 50 μL, ie 5
μL of 10 × PCR reaction buffer, 1 μL of dNTP (10 mM each), 2.2 μL
Mg (OAc) ₂ (25 mM), 1 μL AP1 (10 μM), 1 μL GS
P1, 1 μL of Advantage Genomic Polymerase Mix (50
×), and 37.8 μL of water. The following two-step cycle parameters were used. (Step 1) Seven cycles of denaturation at 94 ° C for 25 seconds, annealing and extension at 72 ° C for 4 minutes. (Step 2) 32 cycles of denaturation at 94 ° C for 25 seconds, annealing and extension at 67 ° C for 4 minutes. After the second stage cycle, the sample was held at 67 ° C. for 4 minutes. The second PCR was performed using the first PC except that AP2 and GSP2 were used.
The reaction was performed under the same reaction conditions as in the R reaction. In addition, each of the used molds is the first P
It was 1 μL of a 50-fold dilution of the CR reaction. The two-step cycle was similar to the first PCR reaction except that the first step was 5 cycles and the second step was 22 cycles.

Oocyte Electrophysiology-cRNA transcript was synthesized from pT-Adv cDNA template linearized with BssHII using T7 RNA polymerase (Ambion). In defolliculated Xenopus laevis, 25 ng of pT-Adv.c
RNA was injected. Three to five days after injection, two-electrode voltage clamp recordings were performed using a Warner OC-725C amplifier (Warner Instruments Corporation, Hamden, CT). The data obtained was analyzed using the pulse / pulse fit software (HEKA, Rambrett / Falz, Germany). The bath solution contained: Adjusted to pH 7.4 with 40 mM Ca (OH) ₂ , 50 mM NaOH, 2 mM TEA-Cl, 1 mM KOH, 0.1 mM EDTA and 5 mM Hepes, methanesulfonate. Boltzmann fit was calculated using a prism (graph pad). Unless stated otherwise,
Results are shown as mean ± standard deviation.

Electrophysiological recordings of β-cells-Whole cell recordings were performed by the standard “Gig-Seal” patch clamp method (Hamil et al.). This whole cell recording pipette is a hemocapillary (Warner)
And pulled with a two-stage puller (PC-10, Narishige International, New York, NY) and a micro-heater (before use).
(MF200-1, World Precision Instruments, Sarasota, FL). Pipette resistances ranged from 2 to 5 MΩ in the internal solution. This recording was performed at room temperature (22 to 25 ° C.). Current was recorded using an EPC-9 patch clamp amplifier (HEKA) and filtered at 2.9 kHz.
Data was obtained with pulse / pulse fit software (HEKA). The voltage-dependent current was corrected for linear leak and residual capacitance using an online P / n subtraction paradigm.

Drug-Mibefradil ((1S, 2S) -2- [2-[[3- (2-benzimidazolyl) propyl] methyl-amino] ethyl] -6-fluoro-1,2,3,4-tetrahydro -1-isopropyl-2-naphthyl methoxy-acetate-dihydrochloride) is described in J. Am. -P. Dr. Closel (Hoffman Laroche,
(Bersel, Switzerland). This is disclosed in U.S. Patent Nos. 5,892,055, 5,811,557, 5,811,556 and 5,808,088.
Can be synthesized according to the method disclosed in the above publication. U.S. Pat. No. 4,808,605 describes mibefradil compounds suitable for use in the present invention.

Des-methoxyacetyl mibefradil (1S, 2S) which is a free alcohol
2- [2-[[3- (2-benzimidazolyl) propyl] methylamino] ethyl] -6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-
Naphthyl hydroxy hydrochloride) was prepared by alkaline hydrolysis. That is, 14.2 mg of mibefradil hydrochloride was dissolved in a mixture of 4 mL of methanol and 1 mL of a 10 N aqueous sodium hydroxide solution (final concentration of mibefradil was 5 mM). The solution was warmed in a boiling water bath for 10 minutes. After this reaction, mass spectrometry was performed. Upon completion of the hydrolysis as determined from the mass spectrum,
The solution was neutralized with 5M aqueous hydrochloric acid. The slight loss of methanol caused by evaporation during the reaction was corrected by adding water to keep the total volume at 5 mL.

Solution—Extracellular solution used for whole cell Ca ²⁺ current recording was 10 mM CaCl ₂ , 110 mM tetraethylammonium-Cl (TEA-Cl), 10 mM CsCl, 10 mM N-2-hydroxy. Ethyl piperazine-N'-2-ethanesulfonic acid (Hepes), 40 mM sucrose, 0.5 mM 3,4-diaminopyridine, pH 7.3. The intracellular solution contains 130 mM N-methyl-D
Glucamine, 20 mM EGTA (free acid), 5 mM bis (2-aminophenoxy) ethane-N, N, N ', N'-tetraacetate (BAPTA), 10
mM Hepes, 6 mM MgCl ₂ , 4 mM Ca (OH) ₂ , pH was adjusted to 7.4 with methanesulfonate. 2 mM Mg-ATP was included in the pipette solution to minimize loss of L-type Ca ²⁺ current. The extracellular solution through-patch recording sucrose 26 mM, 30 mM of TEA-Cl, 10 mM HEPES, KCl of 5 mM, 2 mM of CaCl _2, MgCl _2, contained pH 7.3. The pipette solution contained CsOH of 65 mM, CSMS of 65 mM, 20 mM sucrose, 10 mM HEPES, 10 mM of MgCl _2, 1 mM of Ca (OH) _2, the pH 7.4.

Analysis by Mass Spectrometry-A / VG for obtaining mass spectra of mibefradil and dm-mibefradil
A 70-250 SEQ instrument (VG Analytical, Manchester, UK) was used in fast atom bombardment (FAB) ionization mode. Cultured I
NS-1 cells were treated with 20 μM mibefradil for different lengths of time under each experimental condition. The cell pellet was collected after washing three times with PBS and resuspended in 0.5 mL of medium for mass spectrometry analysis. 20 μL of the internal standard solution (40 μM verapamil, MW: 454) and 5 μL of glycerol were added to 50 μL of the cell sample, and 4 μL of this mixture was used for FAB-MS. Several positive ion spectra were recorded with a mass range of m / z 750-100, mass resolution of 1000, scan rate of 2 sec / 10. For mibefradil, m / z 496 is the dominant ion (
(M + H) ⁺ , with a low intensity sodiated molecular ion at m / z 518. The concentration of mibefradil and hydrolyzed mibefradil was obtained by comparing the intensity at m / z 496 and 424 with the intensity at m / z 455. For calibration, a 50 μM drug standard solution was subjected to mass spectrometry analysis.

Separation of Cytosolic and Membrane Components—After washing off mibefradil from the bath solution, collect the cells and stir the cells in a solution containing 5% acetic acid / CH ₃ CN to remove the membrane. Destroyed. The mixture was then centrifuged and the supernatant was collected and defined as membrane-free components. The pellet was resuspended in 5 volumes of NaOH (10 N): methanol (1: 7) solution at 37 ° C. for 5 minutes. This mixture was neutralized with 0.5 M HCl and centrifuged. The resulting pellet and supernatant were collected separately.

Statistics-All data are presented as mean ± standard deviation, p values shown are Student's t
Calculated using the test.

[0142] Identification and cloning present invention of Example I Pancreatic T-type calcium channels, a cDNA encoding the T-type Ca ²⁺ channel alpha ₁ subunit derived from INS-1 is a cell line secreting insulin in rats provide. The cDNA has been identified and sequenced. From the sequence of the cDNA, 2288 amino acids (SEQ ID NO: 2) that consists of which and neurons of the T-type Ca ²⁺ channel alpha ₁ subunit (alpha ₁ G) as a protein with a 96.3% identity Is shown. Although the transmembrane domain of the protein is very well conserved, this isoform contains three distinct regions and ten single amino acid substitutions in other regions. Sequencing of rat genomic DNA revealed that it was an alternative splice isoform of α ₁ G. Using specific primers and reverse transcription polymerase chain reaction (RT-PCR), both splice variants were demonstrated to be expressed in rat islets. This isoform from INS-1 was also expressed in brain, neonatal heart and kidney. Functional expression of this α ₁ G isoform in Xenopus resulted in a low voltage activated Ca ²⁺ current. These results provide a molecular biological basis for studying the function of T-type Ca ²⁺ channels in β-cells, where the channels play an important role in diabetes.

The rat insulin-secreting cell line INS-1 (Asphali et al., 1)
The cloning and tissue distribution of the isoform of the T-type Ca ²⁺ channel (α ₁ G-INS) from C.992) is described further below.

I that express high levels of T-type Ca ²⁺ current (Buhttacharzy et al., 1997)
Since the NS-1 to obtain cDNA sequences of voltage-dependent Ca ²⁺ channels, previously cloned alpha ₁ subunit (Steer et al, 1995) conserved amino acid sequence containing the six transmembrane segments present in the repeats III of Based on the above, degenerate primers were designed. A DNA fragment of 433 base pairs (bp) was obtained. Next, cDN
The entire sequence of the channel was obtained using the A-terminal rapid amplification (RACE) method. This full-length cDNA (SEQ ID NO: 1) encodes a protein containing 2288 amino acids (SEQ ID NO: 2).

T-type Ca ²⁺ channel genes obtained from β-cells have been identified as α ₁ G, a neuronal isoform of T-type Ca ²⁺ channel (Perez-Raze et al., 1998) and 96.3.
% Amino acid identity. The four intramolecular homology transmembrane domains of the β-cell T-type Ca ²⁺ channel α ₁ subunit are identical to α ₁ G (except for glycine at 1667), and each repeat has six putative transmembrane regions ( S1 to S6) and a hole forming region (P loop). Another region that is very well conserved is located in the intracellular loop between repeats I and II, where parts of the histidine-rich chain contain β-cells as well as neuronal and cardiac T-type Ca ²⁺ channel genes. ^Is also present in the T-type Ca ²⁺ channel gene derived from E. ^coli . This structure in loop _I-II was not observed in the protein sequence of the known high voltage activated Ca ²⁺ channel.

In addition to one amino acid that differs from α ₁ G, the T cell Ca ²⁺ channel gene from β cells contains three unique regions that differ from the amino acid sequence of α ₁ G. These regions include the N-terminal amino acids (aa1-34 of SEQ ID NO: 2), the intracellular loop I _II-III (aa 971-994 of SEQ ID NO: 2) and the intracellular loop L _III-IV (SEQ ID NO:
2 aa 1570-1588).

The amino acid sequence of the obtained channel has an N-terminal region (aa 1-3 of SEQ ID NO: 2).
4) is completely different from α ₁ G, but the nucleotide sequence of this region is shown in FIG. Identical except for the insertion of the four single nucleotides shown in 1A. The insertion of these four single nucleotides determines the different start codon and codon of the amino acid sequence.

To determine the relationship between α ₁ G and the isoform of the T-type Ca ²⁺ channel obtained from INS-1, Sprague-Dawley rats containing the intron and exon between 4845 and 5256 Sections of the genomic DNA sequence were identified. F
ig. As shown in FIG. 1B, an exon encoding SKEKQMA (SEQ ID NO: 5) of the α ₁ G fragment and an exon encoding the 4869-4922 fragment of the INS-1 mutant were found. This region also contains an 8.5 kilobase (kb) intron sequence. Thus, the T-type Ca ²⁺ channel α ₁ subunit and α ₁ G cloned from INS-1 are alternative splice isoforms of the same gene.

Using this genomic DNA sequence, the difference between two nucleotides between α ₁ G cDNA and the isoform cloned from INS-1 was examined. This data is 1
The genomic nucleotide sequence encoding 667 amino acids is GGC (glycine), which is identical to the α ₁ subunit cDNA cloned from INS-1 and the corresponding residue in α ₁ H, but α ₁ G (GCG, Alanine). It is also noted that there are nine additional single amino acid substitutions in the isoform obtained from INS-1 as compared to α ₁ G. Six correspond to amino acids found at similar positions in α ₁ H. That is, 1088 cysteine, 1667 glycine, 1700 alanine, 1735 aspartic acid, 1812 threonine and 1813 leucine.

Regarding the tissue distribution of T-type Ca ²⁺ channels obtained from β-cells and neurons, the expression of the β-cell T-type Ca ²⁺ channel was found in rat brain, heart and kidney but not in liver. I couldn't. Both α ₁ G and the splice form were detected using RT-PCR in rat islets and INS-1 cell preparations. α ₁ H was not detected.

Using a two-electrode voltage clamp technique, the functional expression of T-type Ca ²⁺ channels obtained from β cells was studied in Xenopus. In a solution containing 40 mM Ca ²⁺ , a family of current traces showing T-type Ca ²⁺ current characteristics was obtained (FIG. 2).
A). The current was gradually activated at -40 mV and peaked at -10 mV. Analysis of the time constants for activation and inactivation are described in FIG. 2B. Voltage-dependent activation (F
ig. 2C) and steady state inactivation (FIG. 2D) were fitted to the Boltzmann equation. V1 / 2 calculated for activation and inactivation, respectively, is -23.8 m
V and -45.6 mV. Activation and inactivation k were 5.3 and -6.0, respectively.

Nucleotide cDNA for rat pancreatic T-type calcium channel (SEQ ID NO:
1) and the sequence of amino acids (SEQ ID NO: 2) were determined. SEQ ID NO: 3 is the nucleotide sequence above the coding region, while SEQ ID NO: 4 includes SEQ ID NO: 2.

EXAMPLE II Characterization of the T-type Calcium Channel for Diabetes Glucose-stimulated insulin secretion is a Ca ²⁺ -dependent process, closing ATP-sensitive potassium channels, depolarizing the voltage-dependent Ca ²⁺ channel And open. At glucose concentrations below 3 mM that do not induce insulin secretion, β-cells are resting electrically with a resting membrane potential of about -70 mV. Depolarization is gradually caused by an increase in exogenous glucose, and the degree changes according to the glucose concentration. Depolarization at glucose levels (> 7 mM) that induces insulin secretion is sufficient to reach the threshold potential (-50 mV) at which electrical activity begins.

A simple model for glucose-stimulated insulin secretion is shown in FIG. Summarize in 12.
The resting membrane potential of β cells is mainly determined by the activity of the K-ATP channel. Elevated plasma glucose stimulates glucose uptake and metabolic rate by β-cells. As a result, intracellular ATP (or the ratio of ATP: ADP) increases and K-
This leads to ATP channel closure and membrane depolarization. As a result, the voltage dependence C
The a ²⁺ channel (T-type and L-type) is activated and electrical activity starts. [Ca ²⁺ ] _i rises due to this increase in calcium influx, resulting in insulin secretion.

Rat and human pancreatic β cells have L-type and T-type Ca ²⁺ channels. The physiological function of T-type Ca ²⁺ channels in β-cell insulin secretion has been demonstrated. These channels promote exocytosis by increasing electrical activity in the cell. Under normal conditions, L-type and T-type Ca ²⁺ channels
During glucose-stimulated insulin secretion, they work together to promote elevation of [Ca ²⁺ ] _i . Overexpression of T-type Ca ²⁺ channels in β-cells is at least partially due to the hyperresponse of insulin secretion to glucose-independent depolarization stimulation in GK rats and rats with NIDDM induced by neonatal administration of streptozotocin. Is the cause. However, over-expressed T-type calcium channels over time eventually increase basal Ca ²⁺ due to their window current properties. Thus, depending on the number of channels and the membrane potential, T-type Ca ²⁺ channels in β cells have two effects.

Pharmacological antagonism of T-type calcium channels is a suitable treatment to reduce both insulin resistance and increased insulin secretion in NIDDM patients.

The etiology of NIDDM is complex, and the progression of the disease occurs in multiple stages. Increased β-cell responsiveness stimulates and initiates the progression of the disease. It is unclear what the activity actually increased in this first phase, and what its triggering mechanism is. It may be an increased secretory response or increased β-cell mass. Clearly, however, there is an increase in β-cell activity detected by both basal insulin levels and high postprandial insulin levels called hyperinsulinemia. The result is phase II insulin resistance, especially in insulin-responsive tissues (muscle, liver, kidney, fat) that function to reduce blood glucose levels. Reduced insulin sensitivity accounts for increased blood glucose. This causes the beta cells to secrete more insulin for replenishment. Full blown NIDDM, hyperglycemia and insulin resistance, characterized by the unavoidable lack of insulin secretion by this vicious cycle, are characteristic of the final stages of the disease process.

Each stage of the disease can be characterized by a change in [Ca ²⁺ ] _i . Each phase can be treated with an antagonist of the T-type calcium channel. Electrical β-cells have two types of voltage-gated calcium channels, L-type and T-type calcium channels. Activated by high voltage, has large integral conductance,
L-type calcium channels that are dihydropyridine sensitive are thought to be the major pathway of calcium influx into beta cells (especially with high voltage depolarization). T-type calcium channels that are activated at low voltages, have small integral conductances, and are insensitive to dihydropyridines maintain basal [Ca ²⁺ ] _i (FIG. 8) and Important to increase electrical activity. T-type calcium channels usually promote insulin secretion in β cells by increasing cellular electrical activity. This regulatory function of T-type calcium channels in insulin secretion is important during phase I before onset of diabetes. Antagonizing these T-type calcium channels will reduce β-cell hyperresponsiveness and the resulting hyperinsulinemia and will reduce the progress of the pathogenic pathway to NIDDM.

Even if hyperinsulinemia and associated insulin resistance had already developed, T
Blockers of type 2 calcium channels are still appropriate treatments. Insulin responsive tissues primarily involved in glucose uptake to restore euglycemia increased basal [Ca ²⁺ ] _i during hyperinsulinemia. Indeed, it is this increased basal [Ca ²⁺ ] _i that drives a decrease in the insulin sensitivity of these tissues, and it is known that most of the insulin responsive tissues express T-type calcium channels. Blockers of T-type calcium channels reduce basal [Ca ²⁺ ] _i and alleviate this reduced insulin sensitivity.

Once NIDDM is expressed, it is characterized by changes in glucose metabolism as a result of the abnormal glucose-stimulated secretory response of the β-cell. Β-cell desensitization to glucose is the major lack of secretion of NIDDM. Under normal conditions, L-type and T-type calcium channels act to cooperatively promote an increase in [Ca ²⁺ ] _i during glucose-stimulated insulin secretion. In NIDDM, this cooperation breaks down, attenuating the increase in [Ca ²⁺ ] _i required for insulin secretion.

[0161] The data herein show that L-type calcium channels increase basal calcium levels.
Shows that it is well regulated (Fig. 9A-9D). Foundation
Increase in experimental calcium decreases L-type calcium current sequentially and induces depolarization
[Ca²⁺]_iSeverely reduces the increase in (Fig. 10 and Fig. 11).
The data herein show that T-type calcium channels are responsible for resting basal [Ca ²⁺ ]_iIs also a major regulator of In addition, increased [Ca²⁺] _i There is no negative feedback regulation of T-type calcium channels by
ig. 9A-9D). Basic [Ca²⁺]_iIncreases glucose-stimulated insulin
Lack of secretion is observed in the GK rat model of NIDDM and in the neonatal strike.
T-type calcium current activity as seen in the leptozotocin-induced diabetes model
Under increased circumstances. In the state where the expression of T-type calcium channel is increased
Thus, the reduction of the basic calcium influx by pharmacological intervention alone
Target [Ca²⁺]_i8 (Fig. 8), and the [Ca²⁺ ]_iInducible inhibition may be reduced.

[Ca²⁺]_iAnd there is a clear link between diabetes. [Ca²⁺]_ioperation
Early abnormalities of insulin initiate parallel impairments in insulin secretion and action.
It is a defect that initiates the complications of diabetes. Subsequent metabolic disorders further [Ca ²⁺ ]_iWorsens homeostasis and progressively reduces the overall health of diabetics
Create a harsh cycle. Therefore, [Ca²⁺]_iPharmacological regulation of homeostasis
An agent is a suitable therapeutic means. Hence the use of T-type calcium channel blockers
Will effectively treat and possibly cure diabetes mellitus.

EXAMPLE III Pharmacology of Mibefradil Action Mibefradil has been shown to exert a potent inhibitory effect on T-type Ca ²⁺ currents in vascular smooth muscle cells. The data herein demonstrate that mibefradil also blocks T-type Ca ²⁺ currents in pancreatic β cells in a conventional whole cell patch clamp configuration. Mibefradil (1 μM) was administered to the recording room at time 0 (FIG. 13).
), Control (no drug) showed "decrease". This figure shows that T-type Ca ²⁺ current is more sensitive to mibefradil than L-type Ca ²⁺ current in pancreatic β cells.

Blockade of T-type Ca ²⁺ channels by mibefradil in β cells is reversible.
FIG. 14 demonstrates the reversibility of blockade of T-type Ca ²⁺ currents by mibefradil. In these experiments, mibefradil or NiCl ₂ a very small volume is delivered close to the recording cells. The drug then spread from the cells. The final concentration in the chamber was 1 nM. This experiment indicates that the inhibitory effect of mibefradil on T-type Ca ²⁺ current in pancreatic β cells is due to a reversible interaction between the drug and the channel protein.

In β-cells, T-type Ca ²⁺ channels were able to mediate small but sustained Ca ²⁺ influx by a unique “window” current at approximately resting membrane potential voltages. Like other voltage-regulated channels, T-type Ca ²⁺ channels open and close in response to potential across cell membranes. This voltage dependency is shown in FIG. FIG. These activation and deactivation curves show the ratio of open channels to closed channels over a range of voltages. Unlike most voltage-gated Na ⁺ or L-type Ca ²⁺ channels, the activation and inactivation curves of T-type Ca ²⁺ channels overlap at a range of low voltages (ie, windows). In other words,
In this voltage range, there are some small T-type Ca ²⁺ channels that remain in a non-inactivated state. FIG. Fifteen data were obtained from experiments performed under 10 mM external Ca ²⁺ conditions that shifted the window current to a positive voltage by about 10 mV due to the surface charge effect of divalent ions on the channel.

The presence of window currents provides negative feedback regulation of [Ca ²⁺ ] _i in β cells. When the cell is under unhealthy conditions, it slightly depolarizes and activates the window current, which increases the basal [Ca ²⁺ ] _i and increases the additional Ca ² by L-type Ca ²⁺ channels. ⁺ May protect cells from influx. This process is reversible when the membrane potential returns to normal resting potential (-70 mV).

[0167]Mibefradil regulates basal [Ca2 ⁺ ] _i in pancreatic beta cells The data herein demonstrate that basal [Ca²⁺]_iT when adjusting
Have demonstrated the role of type 2 calcium currents (FIG. 8). [Ca²⁺]_iIs Ca ²⁺ Ca for unbound (340 nm)²⁺Depending on the fluorescence excitation ratio of the bond (380 nm)
After a direct measurement, this ratio was converted to a calcium concentration. The bath solution is 10 mM
NaCl, 4 mM KCl, 2 mM CaCl_Two, And 2 mM MgCl_TwoIncluding
I had. The basic [Ca that fluctuates with an average of about 150 nM²⁺]_iOne cell showing
In 1 hour, administration of 1 μM mibefradil to the chamber immediately
Cium has dropped. This data indicates that T-type calcium currents were
Basic [Ca²⁺]_iIndicates that it is involved in the regulation of

Mibefradil regulates basal insulin secretion Activation of T-type Ca ²⁺ channels at low voltages near the resting membrane potential of pancreatic β cells
This suggests that the channel is responsible for the Ca ²⁺ influx required for insulin secretion under unstimulated conditions. The NIT-1 cell line was selected to demonstrate the effect of mibefradil on basal insulin secretion. NIT-1 is a cell line derived from β cells of non-obese diabetic mice. This cell line expressed high levels of T-type Ca ²⁺ current. The data herein show that 5 μM mibefradil reduced basal insulin secretion to less than 40% of controls (FIG. 17), indicating that the drug reduced the elevated basal insulin secretion levels seen in the early stages of NIDDM. It shows that it can be reduced.

Spontaneous Increase of [Ca ²⁺ ] _i Demonstrates that T-type Ca ²⁺ channels play an important role in calcium influx under unstimulated conditions and thus regulate basal [Ca ²⁺ ] _i For this purpose, a spontaneous increase in the intracellular free calcium concentration was detected using Fluo-3 AM fluorescence imaging.
NIT-1 cells were cultured in a medium containing 3.3 mM glucose, and 2.5 μM
Was previously mounted on Fluo-3 AM. The number of cells with spontaneously increased calcium was counted and compared to all cells used during the 10 minute observation period. 10 μM NiCl ₂ inhibited 90% of the spontaneous increase in basal Ca ²⁺ .

The cellular mechanism of spontaneous increase of intracellular Ca ²⁺ was examined using epifluorimetry. Under unstimulated conditions, some INS-1 cells showing a transient spontaneous increase in [Ca ²⁺ ] _i, a “calcium spike”, were observed. T in this spontaneous process
The role of type Ca ²⁺ channels was also investigated. In one cell with spontaneous calcium spike activity (FIG. 17), NiCl ₂ (30 μM) immediately reduced the spontaneous calcium spike frequency. This result shows that under conditions of absence of glucose, T-type Ca ²⁺ channels alone or together with L-type Ca ²⁺ channels [C
a ²⁺ ] _i suggests that this is the cause of the transient spontaneous increase. These spontaneous calcium spikes can contribute to basal insulin secretion and basal [Ca ²⁺ ] _i control.

However, neither mibefradil or NiCl _{2 had} any effect on basal [Ca ²⁺ ] _i in all β cells. Relatively high initial basic [C
It was observed that only cells with a ²⁺ ] _i would respond to the T-type Ca ²⁺ channel antagonist (FIG. 18). On the other hand, cells with low initial basal [Ca ²⁺ ] _i did not respond or only weakly responded to the T-type Ca ²⁺ channel antagonist. This result indicates that the antagonist of the T-type Ca ²⁺ channel can selectively act on cells with high basal [Ca ²⁺ ] _i and restore normal by inhibiting the window current. .

Example IV Effect on Pancreatic β Cells T-type Ca ²⁺ may play two pathological roles in NIDDM. At an early stage,
The NIDDM patient exhibits hyperinsulinemia and β-cell hyperexcitability. this is,
It may be due at least in part to the increased activity of T-type Ca ²⁺ channels in β cells. In the more advanced NIDDM stage, the decrease in ATP production was
a2 ⁺ channels, resulting in overexpression and membrane depolarization,
Window currents can be induced in β cells that trigger a chronic elevation of a ²⁺ . This increased basal Ca ²⁺ reduces L-type Ca ²⁺ activity and glucose-induced insulin secretion.

Mibefradil has been shown to prevent and relieve the progression of hyperinsulinemia in rats. This result indicates that the drug is a valuable candidate for early stage treatment of NIDDM and prevention of NIDDM in latent patients.

A series of experiments were performed with INS-1 cells to show that T-type Ca ²⁺ enhances insulin secretion by increasing pancreatic β-cell overall excitability.
Specifically, activation of T-type Ca ²⁺ channels increases the frequency of excitation of the depolarizing spike mediated by opening of L-type Ca ²⁺ channels (FIG. 19A). T
Activation of type 2 Ca ²⁺ channels also reduces the onset time of action potentials induced by above-threshold depolarization (FIG. 19B).

To further demonstrate that T-type Ca ²⁺ currents increase β-cell excitability,
Administration of 100 μM NiCl ₂ effectively blocked the T-type Ca ²⁺ channel. In contrast to control experiments, NiCl ₂ delayed the onset of action potential and reduced the number of action potentials.

To directly demonstrate the role of T-type Ca ²⁺ currents in glucose-induced insulin secretion, INS-1 cells were treated with 11.1 mM glucose and various concentrations of NiC
l ₂ and incubated, it was measured insulin secretion. NiCl ₂ reduced insulin secretion in a dose-dependent manner (FIG. 20A). On the other hand, clonal insulin-secreting cells (HIT-T15, did not consistently show T-type Ca ²⁺ current)
Was not affected by NiCl ₂ (FIG. 20B).

These results indicate that T-type Ca ²⁺ channels play an important role in β-cell excitability, and that T-type Ca ²⁺ channel antagonists (such as NiCl ₂ ) effectively increase β-cell excitability. It shows that it reduces to.

While T-type Ca ²⁺ channels promote insulin secretion by increasing the overall excitability of β-cells, the function of T-type Ca ²⁺ channels is a double-edged sword. Under the condition of T-type Ca ²⁺ channels overexpressed in β cells, the function of the window current becomes dominant, resulting in an increase in basal Ca ²⁺ . High [Ca ²⁺ ] _i can cause impaired insulin secretion by inactivating L-type Ca ²⁺ channels.

L-type Ca ²⁺ channels are set in whole cells patches that are partially inactivated by [Ca ²⁺ ] _{i in the} unstimulated state in β-cells. Within the first 5 minutes, the L-type Ca ²⁺ current “runs up” as it increases in cells.
I do. (FIG. 21). This phenomenon is a universal feature of these cells under the recording conditions used. The pipette solution contained no ATP but high concentrations of the calcium chelators BAPTA and EGTA. This rise does not occur when the pipette solution contains high concentrations of Ca ²⁺ . Instead, a rapid descent occurs. This "rise" phenomenon appears to be due to calcium chelation inside the cell. T-type Ca ²⁺ current does not show this effect.

[0180]Intracellular perfusion patch clamp experiments were performed on basal [Ca ²⁺ ] _i in INS-1 cells. It has been shown to regulate the amplitude of L-type Ca ²⁺ current High concentration of Ca²⁺Perfusion (Fig. 9A) of the solution containing²⁺Current
Causes a substantial reduction. L-type Ca²⁺+10 m from the holding potential of -80 mV
Induced by a stepped voltage up to V. This [Ca²⁺]_iThe fura-2 fluorescence ratio
It was measured directly using a meter. The effect of high [Ca²⁺]_i(272 nM
The effect of FIG. 9B. High [Ca²⁺]_iPerfusion realizes the component of high voltage current
It reduces qualitatively, but does not affect low current components. This high [Ca²⁺]_iIs the peak
Causing a shift of the current to a negative voltage,²⁺The current increased at negative voltage. This effect is
As a result, the T-type Ca²⁺It appears to result in an increased current (Fig. 9D). T type C
a²⁺Slow deactivation of current is high [Ca²⁺]_iShows a shift in activation during perfusion
Was. This may explain the shift in IV. Various concentrations of [Ca²⁺]_iIs L type
Ca²⁺The activity of the channel was regulated (Fig. 9C). Existing high [Ca²⁺]_iOr
Low [Ca²⁺]_iPerfusion (632 nM to 0 nM) with time, L-type Ca²⁺Electric
Flow increased, but high [Ca²⁺]_iPerfusion (from 0 nM to 272 nM and 0 nM
632 nM) over time with the L-type Ca²⁺Inhibits current. Therefore, [Ca²⁺]_iof
Level is L-type Ca²⁺Current and T-type Ca²⁺It has a regulating effect on both currents, [Ca ²⁺ ]_iIs the L-type Ca²⁺Make effective feedback adjustments to the current.

[0181]Effect of basic [Ca ²⁺ ] _i on Ca ²⁺ influx Ca²⁺Basic effect on influx [Ca²⁺]_iEffect of Ca²⁺Fu, a pigment indicator
It was examined using ra-2 and fluorescence measurements. Voltage-dependent Ca in one cell²⁺Flow
Inlet perfuses the recording chamber with an osmotic equilibration solution containing 50 mM KCl.
Was obtained. [Ca²⁺]_iIncrease in voltage dependence of nifedipine-sensitive Ca ²⁺ Occurs first through the channel. Static basis in INS-1 cells [Ca ²⁺ ]_iWas about 60-80 nM under the experimental conditions. [Ca²⁺]_iIs fura
-2 Standard curve obtained from calcium imaging kit (Molecular Probes)
Determined by Fruit obtained for calcium binding to fura-2 in this system
The experimental Kd was 296 ± 20 nM. Basic [Ca²⁺]_iStays low
The next voltage stimulation with 50 mM KCl is a rapid and massive flow of calcium into the cells.
When induced repeatedly and stimulated repeatedly when basal calcium is restored, these
Are fixed (Fig. 10). In this experiment, 50m
After M KCl depolarization, cells were repolarized by perfusion of the original 5 mM KCl solution.
. After repolarization, the basic [Ca²⁺]_iGradually recovers, then a second 50 mM KC
l are similar [Ca²⁺]_iA transient current was induced. Basic calcium recovers
If not, a lack of second voltage-induced calcium transients will occur (FIG. 1).
1). In this experiment, after repolarization, the basic [Ca²⁺]_iReturn to its original value
Before the second depolarization was activated [Ca²⁺]_iTransient current is substantially reduced
. These findings are fundamental [Ca²⁺]_iIs voltage-dependent C in INS-1 cells
a²⁺It suggests that it plays an important role in regulating influx. Therefore, the basic [Ca ²⁺ ]_iEffectors can have a significant effect on the amount of calcium that can enter cells.
And

[0182] high basal induced by streptozotocin [Ca ^2+] _i is fundamental in the voltage stimulated Ca ²⁺ influx inhibiting Ca ²⁺ influx Ri by the KCl stimulation [Ca ^2+] Importance of _i a To restate, basal [Ca2 ⁺ ] _i in INS-1 cells was artificially increased by pretreating the cells with the toxic agent streptozotocin. Streptozotocin is DNA
It is known to induce chain breakage, but it has also been shown to induce Ca ²⁺ channel activity in β cells. This data shows that pretreatment of cells with 5 mM streptozotocin for 1 hour and recovery after 3 hours results in a 2-fold increase in basal calcium (FIG. 22). When stimulated with 50 mM KCl, these cells had reduced calcium influx compared to control cells.

[0183]Example V Inhibition of T-type calcium channels by mibefradil metabolites Mibefradil (Ro40-5967) is T-type Ca²⁺Selective inhibitory effect on current
At a high concentration and at a high voltage²⁺Can antagonize current. C
a²⁺The effect of mibefradil on channels is dependent on use and steady state
And L-type Ca²⁺The binding site of mibefradil to the channel is dihydropyridine
Is different from the binding site. Use regular whole cell patch clamps and penetrating patch clamps
By using it, mibefradil provides T- and L-forms in insulin secreting cells.
Ca²⁺It is shown to have an inhibitory effect on both currents. However, L-type Ca ²⁺ The effect on current is time-dependent and less reversible in penetration patch experiments
There was no. Using mass spectrometry, mibefradil is trapped inside cells and
Furthermore, it was proved that a metabolite of mibefradil was detected. Cells of this metabolite
Inside application is the L-type Ca²⁺Selectively inhibited the current, whereas mibefradil was not effective.
Did not. This study shows that mibefradil penetrates into cells and that L-type Ca²⁺Channel
Hydrolyzes into metabolites that specifically block the channel by acting inside
Indicates that it will be decomposed.

T-type and L-type Ca²⁺Confirmed dose-dependent inhibition of mibefradil on both currents
To stand, a whole cell patch clamp and bath perfusion system were used first. The membrane is -90
When the voltage is maintained at -30 mV, the T-type Ca²⁺The current was measured and the film was found to be -40.
mV, the L-type Ca at +20 mV²⁺The current was measured. these
The current was measured twice at each concentration of mibefradil with a measurement interval of 2 minutes. T type C
a²⁺Dose-dependent inhibition of current is described in FIG. 3A. 50% inhibitory concentration (IC₅₀) Is
It was 865 nM. No time-dependent inhibition was observed. In contrast, L-type Ca ²⁺ Inhibition of the current failed to fit a one-to-one binding curve (FIG. 3B). 1 μM
Administration of L-type Ca to 70% of the starting amplitude after 10 minutes.²⁺Current
Gradually decreased (n = 4). This is because a more complex pharmacologic mechanism is that the L-type Ca²⁺Current
Was shown to be involved in the action of mibefradil.

[0185] Next, using the drug diffusion system to test the reversibility of the antagonism of T-type Ca ²⁺ current and L-type Ca ²⁺ current by mibefradil. A small volume (about 2 μL) of drug was delivered in close proximity to the recording cells using a quartz capillary positioned by a micromanipulator. After administration, the drug diffused throughout the recording room containing 2 mL of bath solution. This drug diffusion system was used to test the reversibility of 30 μM NiCl ₂ in T-type Ca ²⁺ currents (FIG. 4). The amplitude of the T-type current decreases rapidly to 40% and 3
Within minutes, it gradually returned to 80% of the initial level. Using this system, the inhibition of T-type Ca ²⁺ current by mibefradil was found to be clearly reversible. In contrast, inhibition of L-type Ca ²⁺ current was not very reversible (FIG. 4).

Poor reversibility and time-dependent inhibition of L-type Ca ²⁺ currents by mibefradil suggested that this drug could have a cumulative effect over time. This hypothesis was tested by applying a very low dose of mibefradil to the cells and recording the L-type Ca ²⁺ current over time in a penetrating patch clamp configuration. FIG. As shown in 5A, 10n
Twenty-five minutes after the administration of M mibefradil, the relative current was reduced to 70%, while the control patch remained unchanged. Incubation of the cells with 10 nM mibefradil for 2 hours further reduced the current density recorded by the penetrating patch (FIG. 5B). At a concentration of 10 nM, mibefradil had no long-term effect on the T-type Ca ²⁺ current.

To test the hypothesis that mibefradil can penetrate from the cell membrane into the cytoplasm and be trapped inside the cells, the presence of mibefradil was examined using mass spectrometry in cells preincubated with 20 μM mibefradil. After washing three times, mibefradil (having a peak at 496 MW) was still detected intracellularly (FIG. 6B).
After one minute incubation, the concentration of intracellular mibefradil was 3.18 ± 0.7.
It was 8 μM (n = 3). After lysis of the cells, the localization of mibefradil in the cells was determined by measuring the concentration of mibefradil in the pellet and the supernatant. The majority (92%) of mibefradil was detected in the supernatant and 0% in the pellet after washing the cells with methanol. This indicates that mibefradil was captured in the cytoplasm. Further, a peak (MW = 423) representing des-methoxyacetylmibefradil (dm-mibefradil), which is a hydrolyzed metabolite of mibefradil, was detected. The dm-mibefradil is the major metabolite as previously described (Wiltshire et al., 1992). By varying the time of the pre-incubation, dm-mibefradil was found to accumulate inside the cells in a time-dependent manner (Fig. 6A). This accumulation is consistent with the idea that dm-mibefradil has a lower membrane permeability than its precursor mibefradil.

[0188] Next, mibefradil or dm- mibefradil tested whether inhibiting L-type Ca ²⁺ current or T-type Ca ²⁺ current from the inner cell. 1 μM mibefradil or dm
-When mibefradil was included in the pipette solution, both L-type and T-type currents were measured in a whole cell patch clamp configuration. FIG. 7A and FIG. As shown in 7B, whereas intracellular applications mibefradil of 1μM it did also have an inhibitory effect on any of the L-type Ca ²⁺ current or T-type Ca ²⁺ current, dm- mibefradil the same concentration the L The type Ca ²⁺ current was specifically blocked. In this series of experiments, the inhibitory effect of dm-mibefradil is thought to be acting on the internal domain of the L-type Ca ²⁺ channel since the bath solution does not contain any drug.

The inhibitory effect of dm-mibefradil on T-type Ca ²⁺ current was similar to its effect when mibefradil was applied to the bath solution. This suggests that the methoxyacetyl group of mibefradil does not play an important role in binding the T-type Ca ²⁺ channel protein to the extracellular receptor site. However, this methoxyacetyl group is required to block L-type Ca ²⁺ channels from inside the cell.
This indicates that modification of the methoxyacetyl group of mibefradil may result in a more selective antagonist of the T-type Ca ²⁺ channel.

[0190]Example VI LVA · Ca ²⁺ current mediates cytokine-induced pancreatic β-cell death Insulin-dependent diabetes mellitus is characterized by selective destruction of pancreatic beta cells
. Long-term treatment with cytokines results in low voltage activated (LVA) Ca in β cells of mice. ²⁺ A current was induced. Concomitant with this basal cytoplasmic free Ca²⁺Concentration ([Ca²⁺] _i ) Was associated with DNA fragmentation and cell death. LVA ・ Ca²⁺Channe
Antagonists of this basic [Ca²⁺]_iIncrease of DNA and DNA fragmentation,
The rate of cell death was reduced. Contact with cytokines affects glucagon-secreting α cells
Ca²⁺Current or basic [Ca²⁺]_iDid not affect the appearance. Therefore
, LVA ・ Ca²⁺Ca through channel²⁺Increased signal is cytokine-induced
It may be an important feature in sex beta cell destruction.

Voltage-sensitive Ca in primary cultures of mouse islet cells²⁺Long term site for current
The effect of Cain treatment was investigated. IL-1β (25 U / mL) and IFNγ (300
U / mL) for 6 hours, and then the cells were treated with LVA.Ca²⁺Induced current
(Fig. 23A). This current is equivalent to 48% of murine islet cells treated with cytokines.
Existed. When cells were treated with either IL-1β or IFNγ alone,
Was not observed. LVA · Ca induced by cytokines ²⁺ The experiment was performed while changing the current recording time. This result is even after 48 hours of treatment
Shows that no further increase in current density occurs. This LVA current is unprocessed
It was not observed in physical cells. Cytokine-induced LVA / Ca²⁺Steady state of current
Inactivation curve is similar to the inactivation curve of LVA current in NOD mouse islet cells.
Similar low voltage characteristics were shown (Fig. 23E). This current is NiCl_Two(10 μM
N = 4; FIG. 23F). Low concentration of NiCl_TwoIs LV
It has been reported to selectively block A current²⁺Large in current density
A significant increase was observed over a voltage of -20 mV to 20 mV. These high voltages
Activated Ca²⁺The current was nifedipine-sensitive current (complete with 10 μM nifedipine).
And the increase in current density causes β-cells in the serum of IDDM patients.
Similar to the increase in L-type calcium current density observed after treatment.

Since α cells are more resistant to the toxic effects of cytokines than β cells, the effect of cytokines on the Ca ²⁺ current in the glucagon secreting cell line (α-TC1) was also examined. This cell line, like α cells, is more resistant to the cytotoxic effects of cytokines. Treatment of α-TC1 cells with IL-1β and IFNγ did not induce LVA · Ca ²⁺ current and did not change the current density (Fi
g. 23C and FIG. 23D). Thus, the induction of LVA Ca ²⁺ current and the increase in Ca ²⁺ current density observed after long-term treatment with cytokines were shown to be β-cell specific.

The LVA.Ca ²⁺ channel is activated at a low membrane potential. This unique feature may then regulate [Ca ²⁺ ] _i under non-stimulated conditions. In fact, the basal [Ca ²⁺ ] _i in cells treated with cytokines was about 3-fold higher than in untreated cells (FIG. 2).
4A). This increase in basal [Ca ²⁺ ] _i was blocked by NiCl ₂ (10 μM), while nifedipine (10 μM), an antagonist of the L-type Ca ²⁺ channel,
) Were not blocked. Cytokines did not increase basal [Ca ²⁺ ] _i in α-TC1 cells (FIG. 24B). These results indicate that LV
A suggests that Ca ²⁺ influx through A · Ca ²⁺ channels is responsible for cytokine-induced increases in basal [Ca ²⁺ ] _i in β cells.

Cytokines have been shown to induce apoptosis in human pancreatic islet cells.
Apoptosis is also a mode of progression of IDDM in NOD mice and cell death in low-dose streptozotocin-induced multiple IDDM in these mice and is involved in β-cell destruction. As one marker of apoptosis, DNA fragmentation prior to lysis of β cells has been reported.

To demonstrate the role of LVA Ca ²⁺ channels in cytokine-mediated DNA fragmentation, β-TC3 cells, a mouse β cell line, were used. At first,
The LVA · Ca ²⁺ current density before and after the cytokine treatment was examined. LVA · Ca ²⁺ current (at V _m = −30 mM) in β-TC3 cells was determined by cytokines (25 U
/ ML IL-1β, 100 U / mL IFNγ, and 100 U / mL TNF
After treatment with α) for 25 hours, it increased from 1.86 ± 0.33 (pA / pF; n = 30) to 3.45 ± 0.47 (pA / pF; n = 10). This indicates that LVA · Ca ²⁺ current in β-TC3 cells is regulated by cytokines as seen in mouse islet cells. FIG. As shown in FIG. 24, cytokine-induced DNA fragmentation showed ladder-shaped oligonucleosome fragments. Three L
The VA.Ca ²⁺ channel blockers NiCl ₂ , amiloride, and mibefradil all independently inhibited cytokine-induced DNA fragmentation. In contrast, nifedipine had no inhibitory effect on DNA fragmentation induced by cytokines. This experiment was performed using β-TC3 cells (n = 2) and β from NOD mice.
Repeat with NIT-1 cells of the cell line (n = 3) with identical results.

Next, the function of LVA · Ca ²⁺ channel in cytokine-mediated cell death of β-TC3 cells was examined. When the medium contains 25 U / mL IL-1β, 100 U / mL IFNγ, and 100 U / mL TNFα, many cells died,
NiCl ₂ (20 μM) effectively reduced the β-cell killing ability of cytokines in both a time-dependent and dose-dependent manner (FIG. 25A and FIG. Respectively).
. 25B). In contrast, nifedipine had no protective effect. Similar results were obtained from experiments performed with mibefradil-treated NIT-1 cells, which also reduced cytokine-induced β-cell death. These results demonstrate that LVA · Ca ²⁺ channels enhance the susceptibility of β cells to the cytotoxic effects of cytokines.

Although preferred embodiments have been described and described in detail herein, various changes, additions, substitutions, and the like can be made without departing from the spirit of the invention, and these are within the scope of the invention. Will be apparent to those skilled in the art.

References Asphali, M. Endocrinology 130: 167-178 (1992). Bayer, E. A. Meth. Enzym. 62: 308 (1979). Bhattacharzy, A. Et al., Endocrinology 138: 3735-3740 (1997).

Void, A. E. III, Current Concepts, The Up John Company, Kalamazoo, Michigan (1991). Campbell, A. M. , Monoclonal Antibody Technology: Experimental Methods in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984). Kapetsch, M. Cell 22: 479-488 (1980).

[0200] Cutterall, W. A. Science 242: 50-61 (1988). Cutterall, W. A. Science 253: 1499-1500 (1991). Komzinsk, P. Et al., Anal. Biochem. 162: 156-157 (1987).

Chrisei, L. Et al., Antisense Research and Development 1 (1): 57-63 (199
1). Christopher Sen, Earl. E. And Marl, Jay. Jay. , Journal of
Medicinal Chemistry 38 (12): 2023-2037 (1995). Davali, A. M. Et al., J. Endocrinology 150: 195-203 (1996).

Ingvar, E. Et al., Immunol. 109: 129 (1972). Gording, Jay. Wu. J. Immunol. Meth. 13: 215 (1976). Han, El. Natl. Acad. Sci. USA 88: 4313-4317 (1991).

Hilliart, M. And Matteson, Dee. R. , J. Gen. Physiol. 91: 1
45-159 (1988). Innis et al., PCR Protocol, Academic Press, San Diego, CA (1990). Kato, S. Et al., Metabolism 43: 1395-1400 (1994).

Kato, S. J. Clin. Invest. 97: 2417-2425 (1996). Care hey, H. H. Diabetes 38: 188-193 (1989). Klein, tea. M. Et al., Nature 327: 70-73 (1987).

Lutz et al., Exp. Cell Res. 175: 109-124 (1988). Mannino, Earl. Jay. And Gould-Foghelite, S. , BioTechnique
s 6: 682-690 (1988). Miller, El. Kei. Bioessays 11: 91-95 (1989).

Peretz-Raise, e. Et al., Nature 391: 896-900 (1998). Rossi, Jay. Jay. AIDS Research and Human Retroviruses 8 (2):
183-189 (1992). Rossi, Jay. Jay. , British Medical Bulletin 51 (1): 217-225.

Sambrook et al., Molecular Cloning: An Experimental Guide, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). Server, N. Et al., Science 247: 1222-1225 (1990). Seino, S. Natl. Acad. Sci. USA 89: 584-588 (1992).

Shigekawa, Kei. And Dvar, W. Jay. , BioTechniques 6: 742-7
51 (1988). Stia, A. Et al., Ligand and voltage-gated ion channels, 113-151.
Page, Earl. Alan, North, CRC Press, Boca Raton (1995
). Sternberger, Jay. A. Et al., J. Histochem. Cytochem. 18: 315 (197
0).

Estee. Gross et al., J. Immunol. Methods 35: 1-21 (1980). Vague, P. And Moulin, Jay. P. , Metabolism 31: 139-144 (1982)
. Wiltshire, H. R. Et al., Xenobiotica 22: 837-857 (1992).

Vang, El et al., Diabetes 45: 1678-1683 (1996). Jani, GC et al., Mol. Endocrinol. 6: 2143-2152 (1992). These and other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments when read in conjunction with the accompanying drawings.

[Sequence list]

[Brief description of the drawings]

FIG. 1A is α ₁ G-INS at the 5 ′ terminal region (aa ₁ to 67 of α ₁ G) (
1 illustrates a comparison of the nucleotide sequences of 1) and α ₁ G (2). The four inserts are indicated by arrowheads. Upper case ATG represents the start codon for each cDNA. FIG. FIG. 1B depicts the partial rat genomic nucleotide organization between domain III and domain IV. Genomic DNA contains 484 of the cDNA sequence.
Exons (shaded circles) specific to α ₁ G between 5 and 5256 and INS-
Exons (shaded rectangles) specific to the α1 subunit of T-type Ca ²⁺ from Example ₁ were included. The other exons (white rectangles) are identical between these two cDNAs. The bold letters indicate 1667 Gly encoding nucleotides.

FIG. 2A to FIG. 2D is α ₁ G-in Xenopus oocytes
2 illustrates the expression of INS. FIG. 2A illustrates the 40 mM Ca2 ⁺ current evoked by depolarizing the pulse from -60 mV to 40 mV. FIG. 2
B illustrates activation and inactivation time constants measured at test potentials between -30 mV and 30 mV. The time constant of this activation was obtained by fitting the Hodgkin-Huxley equation with the m value defined as 4 (n = 6) to the increasing part of the current (activation). The time constant for inactivation was obtained by a single exponential fit (n = 6). FIG. 2C illustrates the voltage dependent conductance (n = 7) and FIG. 2D illustrates the steady state inactivation (n = 3) of the current appearing in the oocyte. FIG. 2C and Fig
. The 2D holding potential was -80 mV. FIG. The 2D current was measured at −10 mV after changing the pre-pulse potential for 1000 ms. The peak current was normalized to the maximum current and then averaged (error bars represent SE).

FIG. 3A and FIG. 3B illustrates the cumulative dose-response relationship of the inhibitory effect of mibefradil on T-type and L-type Ca2 ⁺ currents. Currents were measured using a whole cell patch clamp configuration. The data of the four experiments were normalized and then averaged ±
Plotted as standard error. FIG. 3A is 1 / (1+ [mibefradil] /
2 illustrates a curve generated by fitting the data using a one-to-one binding curve according to the equation Kd). FIG. 3B is the dose response of L-type Ca ²⁺ current obtained when the perfusion solution contains different concentrations of mibefradil.

FIG. 4 illustrates the reversibility of inhibition of T-type and L-type currents by NiCl ₂ and mibefradil, respectively. Open circles and black circles indicate NiCl ₂ (30 μM 2 μM).
L) and T-type C recorded before and after administration of mibefradil (2 μL of 10 μM)
a ²⁺ current. Open squares represent L-type Ca ²⁺ currents recorded before and after administration of mibefradil (2 μL of 10 μM) using a penetrating patch clamp configuration. The T-type Ca ²⁺ current was measured at −30 mV with a holding potential of −80 mV using the whole cell configuration. Arrows indicate the time at which the drug was delivered. N = 3 for each group of experiments.

FIG. 5A and FIG. 5B shows L-type and T-type C in the through patch configuration.
1 illustrates the long-term effect of mibefradil (10 nM) on a ²⁺ current. FIG. 5
In A, closed circles and open circles represent L-type Ca ²⁺ currents recorded in cells treated with and without mibefradil, respectively. Closed triangles represent T-type Ca ²⁺ currents recorded in the cells after administration of mibefradil. Mibefradil was delivered at time zero. N = 4 for each group of experiments. FIG. In 5B, cells were cultured in medium incubated with or without 10 nM mibefradil for 2 hours. This current density was recorded using a through patch clamp configuration. N = 14 for each group of experiments.

FIG. 6A illustrates dm-mibefradil accumulation in the cells as measured using mass spectrometry. The cells were first treated with mibefradil (20 μM) for the time shown in this figure.
) (N = 3). The inset (FIG. 6B) shows the primary data of mass spectrometry showing peaks at 496 and 424. This corresponds to mibefradil and dm-mibefradil, respectively.

FIG. 7A shows the effect of mibefradil and dm on L-type Ca ²⁺ current from inside the cell.
-Illustrates the effect of mibefradil. n = 8, p <0.01 relative to control value. FIG. 7B shows the effect of mibefradil or dm on T-type Ca ²⁺ current from inside the cell.
-Illustrates the effect of mibefradil. n = 4. All data was collected 5 minutes after formation of the whole cell patch. The pipette solution contained 1 μM of the drug.

FIG. 8 illustrates basal [Ca ²⁺ ] _i measured in INS-1 cells. T
Mibefradil (1 μM), an antagonist of type 2 calcium channels, reduced basal [Ca ²⁺ ] _i in one cell in a glucose-free bath solution. This [Ca ²⁺ ] _i is the emission ratio of Fura-2 · AM (F380 / F340)
And then corrected using a standard solution purchased from Molecular Probes, Inc. (Oregon). FIG. 9A illustrates that intracellular perfusion of a solution containing a free calcium concentration of 272 nM inhibits the L-type calcium current. The current was induced by a step voltage up to +10 mV at a holding potential of -80 mV. FIG. 9B illustrates the effect of perfusion at high calcium concentrations on IV calcium current relationships. Closed circles represent cells before perfusion, open circles represent perfusion of 272 nM free calcium. FIG. 9C illustrates the effect of intracellular perfusion at different calcium concentrations over time on L-type calcium current. Squares represent perfusion from high to low calcium (the intracellular solution contained 632 nM and then perfused with a solution containing 10 mM EGTA), triangles represent perfusion from low to 272 nM calcium, and circles Represents low to 632 nM calcium. FIG. 9D illustrates the effect of high calcium on the T-type calcium channel current. The tail current was induced by a step voltage up to -30 mV in 10 ms.

FIG. 10 illustrates that restoration of basal calcium causes fixation of calcium influx. Cells were perfused twice with 50 mM KCl, with intervening perfusion of the original bath solution to restore membrane potential. FIG. 11 illustrates that increased basal Ca ²⁺ results in a lack of Ca ²⁺ transients. The cells are separated by an intervening perfusion of the original bath solution to restore the membrane potential.
Perfusion with 0 mM KCl was performed twice. A second perfusion occurred before the recovery of about 60 nM of the original basal [Ca ²⁺ ] _i .

FIG. 12 illustrates a model for glucose-stimulated insulin secretion
. FIG. 13 shows that mibefradil (1 μM) is T-type Ca in INS-1 cells. ²⁺ Current and L-type Ca²⁺Figure 4 illustrates interrupting the current. This T-type Ca channel
Relative current is obtained by measuring its gradually deactivated tail current
(N = 8).

FIG. 14 illustrates that mibefradil and NiCl ₂ reversibly block T-type Ca ²⁺ currents in INS-1 cells. The drug was administered to the recording room 180 seconds after the start of recording. N = 3. FIG. 15 illustrates the activation and inactivation curves of INS-1 cells, showing "window current".

FIG. 16 shows the effect of NiCl on basal insulin secretion of NIT-1 cells._Two,
2 illustrates the effects of mibefradil and nifedipine. In this experiment, gluco
The source concentration is 3 mM. FIG. 17 is NiCl, a T-type calcium channel antagonist _Two [30 μM] in one cell in a bath solution without glucose [Ca²⁺ ]_i2 illustrates that the frequency of transient spontaneous increases has been reduced. FIG. 18 [Ca²⁺]_iEffect of 30 mM NiC
l_TwoFIG. Data are “high” initial basic [Ca²⁺]_i(About 100n
M). n = 13.

FIG. 19A and FIG. 19B illustrates that hyperpolarization induced an increase in the number of action potentials and a decrease in onset latency. N = 40. FIG. 20A and FIG. 20B illustrates the dose-dependent effect of NiCl ₂ on insulin secretion. Cells were placed in media containing 11.1 mM glucose. Decrease in starting latency. N = 40. FIG. 21 illustrates the "rise" in the whole cell record.

FIG. 22 is KCl in INS-1 cells treated with streptozotocin
6 illustrates inducible Ca ²⁺ influx. n = 13. FIG. 23A-FIG. 23F illustrates the results of cytokine treatment. LV
A · Ca ²⁺ currents were induced by 6-hour cytokine treatment (IL-1β, 25 U / mL, IFNγ, 300 U / mL) in primary cultures of mouse islet cells, but not α-TC1 cells . The LVA current was induced by a test pulse of -40 mV in islet cells (Fig. 23A), but the current was not detected in α-TC1 cells (Fig. 23C). Islet cells with and without cytokine treatment (
FIG. 23B) and Ca ²⁺ current density-voltage relationships obtained from α-TC1 cells (FIG. 23D). Open circles represent the current density of untreated cells (islet cells are n = 1
0, α-TC1 cells are n = 20), and solid circles represent the current density of the cytokine-treated cells (n = 21 for islet cells, n = 21 for α-TC1 cells). This record is 100
Obtained with voltages ranging from -50 mV to +20 mV for m seconds. All experiments were performed at -80 mV. FIG. 23E is a depolarization (-10 mV) pulse for 10 ms at a holding potential of -80 mV, followed by a hyperpolarization (-100 mV) for 50 ms.
Figure 4 shows steady state inactivation of pulse induced LVA tail current. FIG. 2
3F shows that NiCl ₂ (10 μM) blocked the cytokine-induced LVA Ca ²⁺ current induced by a −30 mV step pulse in islet cells.

FIG. 24A and FIG. 24B illustrates the effect of cytokines on [Ca ²⁺ ] _i in mouse islet cells and α-TC1 cells. FIG. At 24A, basal [Ca ²⁺ ] _i of primary culture of mouse islet cells was approximately 3-fold higher after cytokine treatment. NiCl ₂ (10 μM) prevented the increase of [Ca ²⁺ ] _i , while nifedipine (10 μM) did not. FIG. In 24B, basal [Ca ²⁺ ] _i of α-TC1 cells was not affected by cytokine treatment. Cytokine treatment consisted of IL-1β (25 U / mL) and IFNγ (300 U / mL) for 6 hours. FIG. 25A and FIG. 25B illustrates the effect of NiCl ₂ on cytokine-induced β-TC3 cell death. NiCl ₂ (20 μM) significantly reduced cytokine-induced cell death in both a time-dependent manner (FIG. 25A) and a dose-dependent manner (FIG. 25B) (n = 3). Cytokine treatment
FIG. For 25A, IL-1β (25 U / mL), IFNγ (100 U / mL)
And TNFα (100 U / mL). In 25B, IL-1
It consisted of various concentrations of IFNγ as shown in β (25 U / mL), TNFα (100 U / mL) and 25B. The first dose of 0 represents zero concentration for all three cytokines. The concentration of nifedipine was determined according to FIG. 25A and FIG. 2
Both 5B were 10 μM.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C07K 14/47 C07K 16/18 4B065 16/18 C12N 1/15 4C084 C12N 1/15 1/19 4C086 1 / 19 1/21 4H045 1/21 9/00 5/10 C12Q 1/68 A 9/00 G01N 33/15 Z C12Q 1/68 33/50 Z G01N 33/15 33/53 D 33/50 M 33/53 33/566 33/577 B 33/566 33/58 A 33/577 33/68 33/58 C12P 21/08 33/68 C12N 15/00 ZNAA // C12P 21/08 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, N, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, G H, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZWF terms (Reference) 2G045 AA40 BB20 CB01 CB21 CB26 DA12 DA13 DA14 DA31 DA36 DA54 DA80 DB07 FA20 FB02 FB03 FB05 FB07 GC18 JA20 4 B024 AA01 AA11 BA07 BA80 CA04 CA05 CA09 CA12 CA20 DA01 DA02 DA05 DA11 EA02 EA04 GA11 HA13 HA14 HA17 4B050 CC01 CC03 CC10 DD20 EE01 LL01 LL03 LL05 4B063 QA01 QA05 QA13 QA17 QA19 QQ08 QQ43 QR08QR QRQR QRQR QRQR QRQR QRQR QRQR QRQR QRQR QX02 QX10 4B064 AG27 CA10 CA20 CC01 CC24 DA01 DA13 4B065 AA01X AA58X AA72X AA87X AA91X AA99Y AC14 AC20 BA02 CA24 CA27 CA43 CA44 CA46 4C084 AA13 AA17 NA14 ZC35 4C086 AA01 AAA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA FA74

Claims (60)

[Claims] 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 a deoxyribonucleic acid. 3. The isolated nucleic acid molecule according to claim 2, wherein said deoxyribonucleic acid is cDNA. 4. The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1.
An isolated nucleic acid molecule according to claim 3. 5. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes the amino acid sequence set forth in SEQ ID NO: 2. 6. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid is a ribonucleic acid. 7. The isolated nucleic acid molecule according to claim 6, wherein said ribonucleic acid is mRNA. 8. An antisense nucleic acid molecule complementary to at least a part of the mRNA according to claim 7. 9. A cell comprising the antisense nucleic acid molecule according to claim 8. 10. An expression vector comprising the antisense nucleic acid molecule according to claim 8. 11. The expression vector according to claim 10, which is selected from the group consisting of a plasmid and a virus. 12. A cell comprising the expression vector according to claim 10. 13. A method for reducing pancreatic T-type calcium channel expression in a host cell, comprising introducing the antisense nucleic acid molecule according to claim 8 into a host cell, wherein the antisense nucleic acid molecule comprises the mRNA. A method of inhibiting the translation of a T-type calcium channel in said host cell. 14. A ribozyme having a recognition sequence complementary to a part of the mRNA according to claim 7. 15. A cell comprising the ribozyme according to claim 14. 16. An expression vector comprising the ribozyme according to claim 14. 17. The expression vector according to claim 16, which is selected from the group consisting of a plasmid and a virus. 18. A cell comprising the expression vector according to claim 16. 19. A method for decreasing the expression of a pancreatic T-type calcium channel in a cell, comprising the step of introducing the ribozyme according to claim 14 into a host cell, wherein the expression of the ribozyme in the cell is the cell. A method that results in reduced expression of said pancreatic T-type calcium channel in 20. A cell comprising the nucleic acid molecule according to claim 1. 21. An expression vector comprising the nucleic acid molecule according to claim 1. 22. The expression vector according to claim 21, which is selected from the group consisting of a plasmid and a virus. 23. A cell comprising the expression vector according to claim 21. 24. A method for increasing the expression of a pancreatic T-type calcium channel in a host cell, comprising: introducing the nucleic acid molecule according to claim 1 into the cell; and expressing the nucleic acid molecule in the cell. Resulting in the production of pancreatic T-type calcium channels in said cells. 25. A method of screening a substance for altering the function of a T-type calcium channel, the step of introducing the nucleic acid molecule according to claim 1 into a host cell, wherein the nucleic acid molecule is used in the host cell. Expressing the pancreatic T-type calcium channel encoded by: contacting the cell with a substance; and evaluating the contacted cell to determine whether the substance alters the function of the T-type calcium channel Performing the method. 26. The method of claim 25, wherein said evaluating comprises monitoring expression of T-type calcium channels. 27. A method for obtaining a DNA encoding a pancreatic T-type calcium channel, comprising: a DNA molecule encoding a pancreatic T-type calcium channel, the DNA molecule having the nucleotide sequence shown in SEQ ID NO: 1. Selecting; designing an oligonucleotide probe for pancreatic T-type calcium channel based on SEQ ID NO: 1; exploring a genomic or cDNA library of a microorganism using the oligonucleotide probe; Obtaining a clone recognized by the oligonucleotide probe from the library to obtain DNA encoding a calcium channel. 28. A method for obtaining DNA encoding a pancreatic T-type calcium channel, comprising selecting a DNA molecule encoding a pancreatic T-type calcium channel and having the nucleotide sequence shown in SEQ ID NO: 1. Designing a degenerate oligonucleotide primer based on SEQ ID NO: 1; and using a homologous DN encoding a pancreatic T-type calcium channel in the DNA sample using the oligonucleotide primer in a polymerase chain reaction on the DNA sample.
Identifying A. 29. An isolated nucleic acid molecule encoding a pancreatic T-type calcium channel, wherein the first nucleic acid molecule has at least 90% amino acid identity to the second amino acid sequence set forth in SEQ ID NO: 2. A nucleic acid molecule that encodes an amino acid sequence. 30. A DNA oligomer capable of hybridizing to the nucleic acid molecule according to claim 1. 31. A method for detecting the presence of a pancreatic T-type calcium channel in a sample, wherein the sample is a DNA oligomer according to claim 30, wherein the pancreatic T-type calcium channel is present in the sample. Contacting and forming a complex therewith with the DNA oligomer that hybridizes to any of the above, and detecting the complex, thereby detecting the presence of pancreatic T-type calcium channels in the sample. . 32. The method according to 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 according to claim 33, which is encoded by the nucleotide sequence shown in SEQ ID NO: 1. 35. The pancreatic T-type calcium channel protein according to claim 33, which is encoded by the amino acid sequence shown in SEQ ID NO: 2. 36. The method according to claim 36, wherein the second amino acid sequence shown in SEQ ID NO: 2 has at least 90
An isolated pancreatic T-type calcium channel protein, encoded by a first amino acid sequence having a percent amino acid identity. 37. An antibody or a fragment thereof specific to the pancreatic T-type calcium channel protein according to claim 36. 38. The antibody according to claim 37, wherein said antibody comprises a monoclonal antibody. 39. The antibody according to claim 37, wherein said antibody comprises a polyclonal antibody. 40. A composition comprising the pancreatic T-type calcium channel protein according to claim 36 and a carrier having no effect when mixed. 41. A method for detecting the presence of a pancreatic T-type calcium channel protein in a sample, comprising the antibody or fragment thereof according to claim 37, wherein said pancreatic T is present in said sample.
Contacting the sample with any of the type calcium channel proteins to form a complex therewith, and detecting the complex, thereby detecting the presence of pancreatic T-type calcium channel protein in the sample , Including. 42. The method of claim 41, wherein said antibody or fragment thereof is labeled with a detectable marker. 43. A method of altering insulin secretion by pancreatic beta cells, comprising altering the level of a functional T-type calcium channel in pancreatic beta cells. 44. The method of claim 43, wherein altering the level of a functional T-type calcium channel comprises altering the expression of a T-type calcium channel gene in pancreatic beta cells. 45. The method of claim 44, wherein the step of altering expression of the T-type calcium channel gene comprises the step of contacting pancreatic beta cells with a compound that alters expression of the T-type calcium channel gene. 46. The method of claim 45, wherein said compound is an antisense oligonucleotide targeting a T-type calcium channel gene. 47. The method of claim 43, wherein altering the level of a functional T-type calcium channel comprises contacting pancreatic beta cells with an inhibitor of a functional T-type calcium channel. 48. The method of claim 43, wherein altering the level of a functional T-type calcium channel comprises contacting pancreatic beta cells with a compound that interferes with membrane T-type calcium channel formation. 49. The method of claim 43, wherein the pancreatic beta cells are present in a patient suffering from type II diabetes. 50. A method of treating type II diabetes in a patient, comprising administering to the patient an amount of a compound effective to alter the level of functional T-type calcium channels in pancreatic beta cells of the patient. A method that includes 51. The method of claim 50, wherein said compound alters the level of a functional T-type calcium channel by altering the expression of a T-type calcium channel gene. 52. The method of claim 51, wherein altering the expression of the T-type calcium channel gene comprises contacting the pancreatic beta cell with a compound that alters the expression of the T-type calcium channel gene. 53. The method of claim 52, wherein said compound is an antisense oligonucleotide targeting said T-type calcium channel gene. 54. The method of claim 50, wherein said compound is an inhibitor of a functional T-type calcium channel. 55. The method of claim 50, wherein said compound interferes with membrane T-type calcium channel formation. 56. A method of altering basal calcium levels in a cell, comprising altering the level of a functional T-type calcium channel in the cell. 57. A method of altering the action potential of an L-type calcium channel in a cell, comprising altering the level of a functional T-type calcium channel in the cell. 58. A method of altering the death of pancreatic beta cells, comprising altering the level of functional T-type calcium channels in the pancreatic beta cells. 59. A method of altering the proliferation of pancreatic beta cells, comprising altering the level of functional T-type calcium channels in said pancreatic beta cells. 60. A method of altering the flow of calcium through an L-type calcium channel in a cell, the method comprising altering the level of a functional T-type calcium channel in the cell.