Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

• Biological membranes are themselves generally impermeable to ionic species.
• ions enter cells through regulated pores formed from membrane-associated proteins. Most of these regulated pores are voltage-dependent and are thus able to transduce changes in the transmembrane potential into ion flux.
• Noltage-gated ion channels form a "superfamily" of related proteins (cf. Jan et al., Nature, 345, 672 (1990)). Peculiar to this genus is a high degree of conservation in molecular structure.
• voltage-gated channels are membrane bound glycolsylated proteins formed of many subunits. Large ⁇ subunits form a pore in the membrane that is selective for a given ionic species. Each ⁇ subunit contains four domains (I. II, III, and IN).
• Each channel domain has six putative transmembrane helical segments (S,-S 6 ). In general, the segments within each domain are similar but not identical. Aside from overall structural conservation, certain charged residues within the domains are highly conserved among voltage-gated ion channels (Jan et al., supra; St ⁇ hmer et al.. Nature, 339, 597-603 (1989)).
• T-type calcium currents have been implicated in many neurological and muscular functions. For example. T-type calcium current is associated with cardiac pacemaker activity, pain transmission in the central nervous system, and in other physiological functions. Defects in T-type calcium current have been implicated in cardiac arrhythmia, hypertension, and epilepsy. Given their potential clinical value, the pharmacological properties of calcium channels have been the subject of extensive study. Most such studies have involved L-type channels because, unlike T-type channels, L-type calcium channels are readily purified from cell extracts. For example, L-type calcium channels have been purified using dihydropyridine drugs
• L-type calcium channels e.g., nifedipine
• nifedipine e.g., nifedipine
• Such purified and cloned L-type calcium channels have been used to develop assays for drugs affecting L-type calcium channels (see, e.g.. U.S. Patents 5,429,921 and 5.386,025). While many electrophysiological characteristics of T-type calcium currents are known, the lack of isolated T-type channels has stalled research into the pharmacology and biophysics underlying the T-type calcium current, at least in comparison with other calcium channels.
• the present invention provides an isolated or substantially purified nucleic acid encoding a protein comprising at least one domain of a T-type calcium channel and cells and cell lines expressing such nucleic acids.
• the present invention also provides an isolated or substantially purified T-type calcium channel and an isolated or substantially purified antibody molecule recognizing an epitope on a T-type calcium channel protein.
• Figures 1 A- IE compare the complete amino acid sequences of three types of T-type calcium channels ( ⁇ lG (or Ca,,T.l), ⁇ lH (or Ca.T.2), and all (or Ca.T.3)), indicating conserved functional domains.
• Figures 2A-2D are graphic representations of the current- voltage relationships of three cloned T-type calcium channels ( Figures 2A, 2B, and 2C) and a cloned R-type calcium channel ( Figure 2D).
• Figure 3 A is a graphic representation of the average current-voltage curve for cloned T-type calcium channels ( ⁇ lG. triangles, ⁇ lH, inverted triangles, all, circles), and a cloned R-type calcium channel (filled squares).
• Figure 3B compares the normalized conductance of a cloned T-type calcium channel at three different concentrations of BaCl 2 .
• Figure 4 depicts average kinetics of the tail current as a function of repolarization potential for ⁇ lG (triangles), ⁇ lH (inverted triangles), all (circles), and a cloned R-type calcium channel (filled squares).
• Figures 5 A and 5B graphically present data concerning the use of a cloned T- type calcium channel to detect drugs affecting the channel.
• Figure 6A depicts the effect of 100 ⁇ M on current-voltage relationships with a single dosage of miberfradil.
• Figure 6B illustrates the effect on T-type channel conductance of various doses of miberfradil.
• the present invention provides an isolated or substantially purified nucleic acid encoding a protein comprising at least one domain of a T-type calcium channel ⁇ subunit.
• the nucleic acid can be of any type, and it can include other elements aside from a sequence encoding a T-type calcium channel domain or domains.
• the nucleic acid comprises RNA. it can also include regulatory sequences suitable to permit translation of the RNA.
• an RNA nucleic acid of the present invention preferably has at least one ribosome entry site, and preferably has a polyadenosine tail for stabilizing the RNA in the cellular environment.
• DNA nucleic acids of the present invention can have regulatory elements for promoting the transcription of sequence encoding the T-type calcium channel into an RNA such as that described above.
• a DNA nucleic acid of the present invention can have a promoter and/or an enhancer sequence.
• the nucleic acid can be any type of nucleic acid, the nucleic acid preferably comprises a cDNA.
• a cDNA nucleic acid is preferred over other nucleic acids to permit the nucleic acid to be readily cloned, sequenced, and expressed in a wide variety of cells. The choice of promoter and/or an enhancer will largely depend on the milieu in which the nucleic acid is to be expressed.
• the regulatory elements are bacterial promoters.
• the regulatory elements are able to effect expression in mammalian cells. While many such regulatory elements are known in the art, examples include prokaryotic promoters and viral promoters (e.g., retroviral ITRs, LTRs, immediate early viral promoters (IEp), such as herpesvirus IEp (e.g., ICP4-IEp and ICPO-IEp), cytomegalovirus (CMV) IEp, and other viral promoters, such as Rous Sarcoma Virus (RSN) promoters, and Murine Leukemia Virus (MLN) promoters).
• IEp immediate early viral promoters
• CMV cytomegalovirus
• RSN Rous Sarcoma Virus
• MSN Murine Leukemia Virus
• promoters are eukaryotic promoters, such as enhancers (e.g., the rabbit ⁇ -globin regulatory elements), constitutively active promoters (e.g., the ⁇ -actin promoter, etc.). signal specific promoters (e.g., inducible promoters such as a promoter responsive to RU486. etc.), and tissue-specific promoters (e.g., those active in epidermal tissue, dermal tissue, tissue of the digestive organs (e.g..).
• enhancers e.g., the rabbit ⁇ -globin regulatory elements
• constitutively active promoters e.g., the ⁇ -actin promoter, etc.
• signal specific promoters e.g., inducible promoters such as a promoter responsive to RU486. etc.
• tissue-specific promoters e.g., those active in epidermal tissue, dermal tissue, tissue of the digestive organs (e.g...
• the isolated or substantially purified nucleic acid of the present invention encodes all or part of a T-type calcium channel ⁇ subunit.
• a "calcium channel” includes a protein structure for facilitating the flux of calcium ions across a biological membrane into which the calcium channel is inserted.
• a "T-type channel' " is a type of voltage- gated ion channel that facilitates the flux of ions when the membrane potential of a biological membrane into which it is inserted experiences a slight depolarization.
• a T-type calcium channel can begin to gate from about -60 mV to about -30 mV (i.e.. about -45 mV to about -35 mV) in about 10 mM Ba 2+ . Additionally.
• an amino acid sequence of a T-type calcium channel can vary from those listed, and it is within the state of the art to change a nucleotide sequence encoding a T-type channel to introduce mutations into the protein.
• mutations comprising insertions or deletions can be introduced on either the amino- or carboxy-terminus of the protein, or such mutations can be intrasequence insertions or deletions.
• the isolated or substantially purified polynucleotide can itself be employed as a probe to screen a library as described to isolate a second nucleic acid.
• one of the polynucleotides will be complementary to a portion of the sequence encoding the T-type calcium channel, and the other isolated nucleic acid will be "sense.”
• one of the two isolated polynucleotides (the "sense " strand) itself encodes a T-type calcium channel, or at least one domain thereof.
• Such a sequence can be cloned to be operably linked to suitable regulatory elements, as described, to produce a T-type calcium channel.
• the nucleic acids of the present invention are also useful for isolating other sequences encoding T-type calcium channels, or derivatives thereof.
• the vector can be introduced into the cell in any manner suitable for the cell type and vector employed.
• the vector can be used to prepare an RNA transcript in vitro (e.g., a capped cRNA) which is then introduced into the host cell by standard methods (such as injection). Such techniques are preferred when the host cells do not actively transcribe DNA (such as oocytes).
• a DNA vector is introduced into the cell such that it is transcribed within the cell.
• the vector can be introduced into the cell such that it forms an extrachromosomal segment of genetic material in the cell, as is the case with many types of viral vectors.
• the vector can introduce the nucleic acid into the chromosomal DNA of the host cell.
• the method involves first expressing a T-type calcium channel in a cell to produce an active channel, as herein described.
• the cell expressing the channel is then exposed to a solution containing a putative drug for interfering with the channel. Thereafter, the presence or absence of calcium flux in response to a change in membrane potential is assayed.
• Any such assay can be employed within the context of the present invention, (e.g.. using labile dyes, radioisotopes (e.g.. 43 Ca). recording electrophysiological changes in the membrane, etc.).
• a quick method of assaying for calcium flux is first to introduce a calcium-sensitive labile dye into the cells.
• the vector is introduced into myocardial cells.
• the vector is introduced into neurons (e.g.. thalamic neurons).
• the nucleic acid within the vector is expressed to produce active T-type calcium channel.
• an nucleic acid having a sequence antisense to a sequence encoding a T-type calcium channel (or a portion thereof) can be expressed within a cell. The presence of an antisense sequence can down-regulate the expression of native T-type calcium channel genes by hybridizing to T-type channel mRNA within the cell.
• the present invention is useful to treating disorders associated with over-expression of T-type calcium channels.
• antibodies directed to T-type calcium channels are potential reagents for studying the channels as well as for therapy.
• Such antibodies can be produced by any suitable method, many of which are well known in the art.
• the antibodies can comprise polyclonal antisera obtained from innoculated animals.
• the antibody molecules can be monoclonal antibodies obtained from a cell line (e.g., a hybridoma cell line).
• a cell line e.g., a hybridoma cell line.
• the present invention provides a cell which produces such antibodies.
• Such a cell can be in vitro or in vivo: however, where the cell is in vitro, preferably it is within an established cell line consisting essentially of such cells.
• capped cRNA was injected into the oocytes in a volume of 50 nl.
• oocytes were injected with 100 ng capped cRNA and incubated for one week prior to assay.
• Cells were voltage clamped using a two-microelectrode voltage clamp amplifier as described (Bernal et al., J. Pharmacol. Exp. Ther., 282, 172-80 (1997)).
• the standard bath solution contained the following: 40 mM Ba(OH) 2 .
• This example demonstrates the production of cell lines stably expressing the cloned ⁇ l G. ⁇ l H. and al l proteins.
• the internal pipette solution contained the following: 55 mM CsCl, 75 mM CsSO , 10 mM MgCl 2 , 0.1 mM EGTA, 10 mM HEPES, pH adjusted to 7.2 with CsOH.
• the external Tyrodes solution was the following: 140 mM NaCl, 6 mM KC1, 2 mM CaCl 2 , 10 mM glucose, 5 mM HEPES, pH 7.4.
• the recording solution contained the following: 10 mM BaCl 2 solution (or 2 mM CaCl 2 ), 140 mM tetraethylammonium (TEA) chloride, 5 mM CsCl.
• Single channel conductance is complicated by the low probability of channel opening at negative potentials when the driving force is large. Thus, single channel conductance was measured similarly for measurements of tail currents to enhance channel opening at negative potentials.
• Single channels were measured with standard depolarizing bath and pipette (1 15 mM BaCl 2 , 1 mM EGTA, and 10 mM HEPES, pH 7.4) solutions (Lacerda et al., Biophys. J., 66. 1833-43 (1994)). Data were analyzed with TRANSIT (VanDongan, Biophys J., 70, 1303-15 (1996)).

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• 1998
  • 1998-10-30 EP EP98956416A patent/EP1036170A1/de not_active Withdrawn
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