WO1999029847A1

WO1999029847A1 - T-type voltage-gated calcium channels and method of using same

Info

Publication number: WO1999029847A1
Application number: PCT/US1998/023161
Authority: WO
Inventors: Edward Perez-Reyes; Leanne L. Cribbs
Original assignee: Loyola University Of Chicago
Priority date: 1997-12-05
Filing date: 1998-10-30
Publication date: 1999-06-17
Also published as: EP1036170A1; CA2312477A1

Abstract

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.

Description

T-TYPE VOLTAGE-GATED CALCIUM CHANNELS AND METHOD OF USING SAME

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Number HL58728 awarded by the National Heart. Lung, and Blood Institute of the National Institutes of Health. The United States Government may have certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to cloned T-type calcium channels.

BACKGROUND OF THE INVENTION Biological membranes are themselves generally impermeable to ionic species.

Thus, 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. Generally, voltage-gated channels are membrane bound glycolsylated proteins formed of many subunits. Large α subunits form a pore in the membrane that is selective for a given ionic species. Each α subunit contains four domains (I. II, III, and IN). Each channel domain has six putative transmembrane helical segments (S,-S₆). 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; Stϋhmer et al.. Nature, 339, 597-603 (1989)).

Differences in charged residues between groups of voltage- gated ion channels confer properties unique to each subgroup, such as ion selectivity. For example, most voltage gated ion channels are selective for either sodium, potassium or calcium. Known calcium channels require a ring of negative charge provided by glutamate residues found at similar locations in each of the domains (Yang et al., Nature, 366, 158-61 ( 1993)). Noltage-gated channels are often classified on the basis of their electrophysiology. The resting membrane potential of most animal cells is between about -70 mV and -80 mV. When the membrane becomes depolarized (moved towards 0 mV), various membrane channels become activated (they are said to "open^"). Thus, one basis for classifying membrane channels is the membrane potential necessary to activate (or ^■^316'') them (voltage dependency). For example. "T-type^" calcium channels are activated at a lower voltage than L- or N-type channels (Nowycky et al.. Nature, 316. 440-43 (1985)). Other physiological properties are the activation kinetics, inactivation kinetics, tail current (deactivation kinetics), and single channel conductance. Thus, in comparison to other calcium currents. T-type calcium current is characteristically short (Chen et al.. J. Gen. Physiol, 96, 603-30 (1990)), and it exhibits characteristically slow activation kinetics near threshold, fast inactivation kinetics, and slow tail current (Randall et al., Neuropharmacol, 63. 879-93 (1997); Carbone et al.. Nature, 310. 501-02 (1984); Nilius et al.. Nature, 316, 443-46 (1985)).

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

(e.g., nifedipine) which can bind with sufficiently high affinity to serve as a ligand for purifying L-type calcium channels. 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. Indeed, while it is generally assumed that voltage-sensitive ion channels are responsible for the current, no such channel protein, nor any nucleic acid encoding such a protein, has been isolated. In view of the foregoing problems, there exists a need for an isolated T-type calcium channel and a nucleic acid encoding a T-type calcium channel.

BRIEF SUMMARY OF THE INVENTION 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.

The present invention is useful for exploring the electrophysiology and pharmacology of the T-type calcium current. Such knowledge can lead to the development of drugs for potentiating or attenuating T-type calcium channels. Thus, the present invention provides an assay for identifying potential drugs affecting T-type calcium channels by exposing cells expressing a T-type calcium channel to a putative drug and then measuring the calcium flux in response to a change in membrane potential. The identification of drugs affecting T-type calcium channels will facilitate even greater understanding of the biophysics of these proteins. Furthermore, some such drugs could have potential clinical applications.

The invention can best be understood with reference to the accompanying drawings and in the following detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A- IE compare the complete amino acid sequences of three types of T-type calcium channels (αlG (or Ca,,T.l), αlH (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 (αlG. triangles, αlH, 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 BaCl₂.

Figure 4 depicts average kinetics of the tail current as a function of repolarization potential for αlG (triangles), αlH (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 μM 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.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

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 α 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. For example, where the nucleic acid comprises RNA. it can also include regulatory sequences suitable to permit translation of the RNA. Thus, 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. Similarly, 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. For example, a DNA nucleic acid of the present invention can have a promoter and/or an enhancer sequence. While 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. Thus, for expression in bacterial cells, the regulatory elements are bacterial promoters. Similarly, for expression in mammalian cells, 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). Other suitable promoters are eukaryotic promoters, such as enhancers (e.g., the rabbit β-globin regulatory elements), constitutively active promoters (e.g., the β-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.. cells of the esophagus, stomach, intestines, colon, etc., or their related glands), smooth muscles, such as vascular smooth muscles, cardiac muscles, skeletal muscles, lung tissue, hepatocytes, lymphocytes, endothelial cells, sclerocytes. kidney cells, glandular cells (e.g., those in the thymus, ovaries, testicles, pancreas, adrenals, pituitary, etc.), tumor cells, cells in connective tissue, cells in the central nervous system (e.g., neurons, neuralgia, etc.), cells in the peripheral nervous system, and other cells of interest).

The isolated or substantially purified nucleic acid of the present invention encodes all or part of a T-type calcium channel α subunit. As used herein, 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. As used herein, 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. Thus, 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 Ba²⁺. Additionally. T-type channels of the present invention exhibit a slow deactivation (tail current) following depolarization. Thus, a T-type calcium channel can exhibit a tail current that decays exponentially with a tau value from about 1 ms to about 10 ms (e.g., from about 4 ms to about 7 ms. such as about 6 ms) following repolarization to a membrane potential from about -80 mV to about -60 mV in a solution with a Ba²⁺ concentration of from about 10 mM to about 40 mM. Another defining characteristic of T-type calcium channels is that they exhibit small single channel conductance. Thus, for example, a T-type channel exhibits a single channel conductance of from about 4 pS to about 12 pS (e.g.. from about 6 pS to about 10 pS), and typically from about 7 pS to about 9 pS in a solution with a Ba²⁺ concentration of about 0.1 M. The isolated or substantially purified nucleic acid of the present invention encodes all or part of any T-type calcium channel having at least one of the aforementioned electrophysiological properties when properly assembled within a cellular membrane. The general structure of calcium channels is summarized above and is otherwise known in the art. Thus, for example, the nucleic acid can encode one of the four functional domains mentioned above. As used herein, a domain of a T- type calcium channel is any protein structure able to associate with three other domains to form a tetrameric body functioning as a T-type calcium channel. While the native T-type calcium channel structure includes all four domains in a single polypeptide (indicated in Figures 1 A- IE), a domain can exist as a polypeptide species separate from those containing the other domains. Such separate domains are able to associate within the plasma membrane to form a functional channel. Alternatively, where a plurality of domains are linked within a common polypeptide. the linkage can deviate substantially from the native linkage. Thus, for example, the domains can be linked by polypeptide sequences other than those sequences linking the domains in the native protein (e.g., non-native polyglutamate linkages). Indeed, the domains themselves can include non-native linkages between membrane-spanning elements within the domains. Aside from these modifications, the nucleic acid can encode a chimeric calcium channel domain (or an entire channel) comprising a portion of a T- type calcium channel and a portion derived from another calcium channel (or other channel) protein. For example, the chimera can include portions of domains from T- type channels responsible for low voltage gating and portions of domains from other calcium channels responsible for slow inactivation. Such a protein exhibiting T-type gating but longer inactivation kinetics would facilitate pharmacological research. As mentioned, nucleic acids of the present invention can encode an entire T- type channel (i.e.. a T-type channel protein comprising four functional domains). It has been discovered that at least three genes encoding T-type calcium channels exist in humans and rats (i.e.. αlG (or Ca,T. l ). αlH (or Ca,T.2). and all (or Ca,T.3)). and alternate splicing of these isoforms exist. Examples of the amino acid sequences of full-length T-type channels, and the sequences of suitable coding nucleic acids are set forth at SEQ ID NOs: l-8 (αl G sequences). SEQ IS NOs:9-10 (αlH sequences), and SEQ ID NOs: 1 1-12 (all sequences). However, the invention is not limited to these exemplary sequences. Indeed, as mentioned, 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. Indeed, for conducting electrophysiological assays, it may be desirable to introduce mutations into such a protein. For example, 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. Where the electrophysiological properties of the calcium channel are to be conserved, such mutations preferably are in regions other than the membrane spanning domains. However, in some applications (e.g.. to decrease inactivation kinetics), the changes can be within the membrane-spanning regions. Moreover, as mentioned above, the sequence can form a protein having only one functional domain of a T-type calcium channel. Additionally, the sequence can also form a chimeric protein or domain, such as those described above.

Aside from insertions and deletion mutations of native T-type calcium channel sequences, a T-type calcium channel can include substitutions of amino acid residues. e.g., for those indicated in SEQ ID NOs: 1-12. Preferably, and especially where such a substitution is within a membrane spanning region, the substitution is conservative. Thus, within membrane spanning domains, positively-charged residues (H, K, and R) preferably are only substituted with positively-charged residues; negatively-charged residues (D and E) preferably are only substituted with negatively-charged residues; neutral polar residues (C. G. N, Q. S. T, and Y) preferably are only substituted with neutral polar residues; and neutral non-polar residues (A. F, I. L, M, P. V, and W) preferably are only substituted with neutral non-polar residues. Preferably, any amino-acid substitution within the membrane-spanning regions does not alter this conservation. Most preferably, any substitution, deletion, or insertion does not alter the IVS4 domain. In each of the exemplary T-type calcium channel α subunit sequences, the putative IVS4 region comprises SEQ ID NO: 13. Given the strong sequence conservation among families of voltage-gated ion channels, it is likely that this sequence or a derivative sequence, will be present in T-type channels. Thus, the present invention provides any T-type calcium channel (or a nucleic acid encoding such a T-type calcium channel) comprising SEQ ID NO: 13 or a sequence derived from SEQ ID NO: 13 having conservative amino acid substitutions, as described above. The nucleic acid of the present invention encoding all or a part of a T-type calcium channel can be isolated via any suitable method. For example, prior to the present invention, one of skill in the art could design a probe based on the sequence of known. non-T-type. calcium channels and use such probe to screen a genetic library. If such a screen were to identify a putative calcium channel, the researcher could then attempt to clone the entire nucleic acid to characterize it. Similarly, prior to the present invention, to isolate a nucleic acid encoding a T-type calcium channel, one of skill in the art could consult publicly available databases containing DNA sequences (e.g., Genbank) to locate nucleic or amino acid sequences representing a portion of a T-type calcium channel protein or nucleic acid. However, such databases contain no sequence for a full-length T-type calcium channel or identify any sequence as a T-type channel. Such methods assume that T-type calcium channels share sufficient sequence identity with known calcium channel nucleic acids to cross-hybridize, an assumption not supported by any published report. Moreover, prior to the present invention, no partial sequence in such databases was identified as corresponding to a T-type calcium channel. Thus, prior to the present invention, the presence of partial sequences in the public DNA databases could facilitate the isolation of T-type calcium channels only with the exercise of a considerable degree of speculation on the part of the researcher.

By providing several sequences pertaining to T-type calcium channels and a comparison presenting conserved regions and domains, the present invention greatly facilitates the isolation of other nucleic acids encoding T-type calcium channels (or derivatives thereof) with much less experimentation. Thus, while any of the methods discussed above can be employed to isolate other members of this genus, preferably, a nucleic acid encoding a T-type calcium channel is isolated by probing a genetic library using a probe that hybridizes to a DNA encoding a peptide sequence contained in (or similar to) a known T-type calcium channel (e.g.. SEQ ID NOs: 1-12). To facilitate the isolation of a T-type calcium channel, the present invention provides an isolated polynucleotide hybridizing to a portion of the nucleic acid of the present invention encoding a T-type calcium channel (or a portion thereof). Thus, for example, the present invention includes an isolated polynucleotide hybridizing to SEQ ID NO: 1-12. The isolated polynucleotide can hybridize to all or any portion of the sequence encoding the T-type calcium channel. To isolate such a polynucleotide. any portion of a sequence encoding a T-type calcium channel can be employed as a probe to screen a genetic library, and such screening can be accomplished by standard techniques known in the art. While the probe can hybridize to any portion of such a DNA. preferably the probe is designed to hybridize to a DNA encoding a polypeptide sequence that is highly conserved among T-type calcium channels but is less conserved between the genus of T-type calcium channels and other proteins. Such peptide sequences are readily apparent from the sequence comparison set forth in Figures 1A-1E. Generally, the specificity of hybridization in a genetic screen varies depending on the length of the probe and the stringency (e.g., temperature, salt and detergent concentration, etc.) of hybridization. Stringency of hybridization is broadly classified as "high.'^' "moderate,^" or "low." and the parameters of these terms are well recognized in the art (see. e.g., Sambrook et al.. "Molecular Cloning, a Laboratory Manual,"' Cold Spring Harbor Press, 1989). The isolated polynucleotide hybridizing to a portion of the nucleic acid encoding a T-type calcium channel can hybridize under any desired stringency conditions. However, for identifying other T-type channels, preferably, the hybridization occurs under moderate stringency, and most preferably under high stringency.

Of course, 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. In such a screen, 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." Preferably, 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. Thus, aside from using the nucleic acid of the present invention 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.

However isolated, the isolated or substantially purified nucleic acid of the present invention is useful, in part, for producing all or a portion of a T-type calcium channel. Thus, the nucleic acid can be introduced into a suitable milieu for driving its expression. Because T-type channels are transmembrane proteins, preferably such a milieu is a living cell. However, it should be understood that the nucleic acid can also be expressed in vitro under conditions, such as those known in the art. suitable for in vitro transcription and translation. However produced, the present invention includes any protein, such as a recombinant protein or an isolated or substantially purified protein, including all or a portion of a T-type calcium channel or a protein derived from a T-type calcium channel. For expression in a living cell, the nucleic acid must be introduced into the cell. As nucleic acids are generally introduced into cells as part of genetic vectors, the present invention provides a vector having a T-type calcium channel nucleic acid of the type described above. Any type of vector suitable for introducing the nucleic acid into a host cell is within the context of the present invention. Examples of such vectors include naked DNA and RNA vectors (such as oligonucleotides, plasmids. capped cRNA, etc.). viral vectors such as adeno-associated viral vectors (Berns et al., Annals of the New York Academy of Sciences, 772. 95-104 (1995)), adenoviral vectors (Bain et al.. Gene Therapy, 7, S68 (1994)). herpesvirus vectors (Fink et al., Ann. Rev. Neurosci., 19, 265-87 (1996)), packaged amplicons (Federoff et al., Proc. Nat. Acad. Sci. USA, 89, 1636-40 (1992)), pappiloma virus vectors, picornavirus vectors, polyoma virus vectors, retroviral vectors, SV40 viral vectors, vaccinia virus vectors, and other vectors. Once a given type of vector is selected, its genome must be manipulated for use as a background vector, after which it must be engineered to incorporate exogenous polynucleotides. Such manipulations are known in the art. The vectors of the present invention are useful for introducing a nucleic acid encoding all or a portion of a T-type calcium channel into a host cell. Thus, the present invention provides a cell into which the vector of the present invention has been introduced. The host cell can be any cell suitable for expressing the nucleic acid (e.g., bacteria, insect cells, mammalian cells, etc.). The host cell can thus be in vitro or in vivo. Preferably the cells do not exhibit native T-type calcium current. A preferred cell type is HEK-293 cells because they contain genetic elements that facilitate the expression of transgenes from a variety of expression vectors. For facilitating electrophysiological recordings, oocytes (e.g., Xenopus oocytes) are preferred, as they are large and readily handled.

The vector can be introduced into the cell in any manner suitable for the cell type and vector employed. In one embodiment, 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). In other embodiments, a DNA vector is introduced into the cell such that it is transcribed within the cell. For example, 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. Alternatively, the vector can introduce the nucleic acid into the chromosomal DNA of the host cell.

Preferably, a cell into which the nucleic acid is introduced is also able to express the nucleic acid to produce the α subunit protein. The expression of the nucleic acid can be detected by probing the cell for the presence of T-type calcium channel mRNA. such as via Northern hybridization analysis, in situ hybridization, etc. More preferably, however, the cell is able to express the nucleic acid to produce the protein including all or a portion of a T-type calcium channel. In such cells, expression of the nucleic acid is confirmed by detecting the protein, for example, by probing cellular extracts with an antibody recognizing the protein (e.g.. on a Western blot, etc.).

In the membrane of the cell producing the protein, the expressed protein contributes to the formation of a functional calcium channel. Where the protein encodes an entire α subunit, the full protein will possess some or all of the electrophysiological properties of T-type calcium channels described above. Where the protein encodes less than an entire channel α subunit (e.g., a domain), the protein will aggregate with other constituent domains in the membrane to form a functional channel. Thus, the presence of the protein can be detected by assaying the cell for T-type calcium channel activity. Indeed, assaying for channel activity serves to determine whether a nucleic acid encoding a putative calcium channel, in fact. encodes a species of T-type channel (as opposed to a member of another genus of calcium channels). For example, when large cells (e.g., oocytes) are used as the host cells, the electrophysiological properties of the channel can be investigated. Thus, the membrane activity of whole cells expressing the nucleic acid can be measured directly, such as via patch clamp techniques using a voltage clamp electrode and a current electrode (Bernal et al.. J. Pharmacol. Exp. Ther., 282, 172-80 ( 1997)). Alternatively, the activity of single channels can be measured, such as with a standard depolarizing bath and pipette solutions (Lacerda et al.. Biophys. J., 66, 183-43 (1994)). However measured, the properties of cells into which the putative nucleic acid is introduced are compared to the channel conductance, voltage dependency, activation kinetics, inactivation kinetics, or tail current known for T-type channels and discussed above. A measure of current density (e.g., pA/pF) can also be used to assess the level of gene expression in the cells, normalizing for cellular volume.

While, in accordance with the present invention, an isolated cell into which the T-type calcium channel nucleic acid has been introduced (and preferably stably expressing the nucleic acid to produce the protein) can be prepared, preferably, such transfection protocols result in a population consisting essentially of such transfected cells. For standardizing the results of many experiments, it is even more desirable to employ an established cell line consisting essentially of such cells. Preferably, for use in high throughput assays, cell lines stably expressing a T-type calcium channel exhibit a current density of at least about 40 pA/pF (e.g.. at least about 45 pA/pF). such as about 50 pA/pF or even 55 pA/pF or higher. Preferably, a cell line in accordance with the present invention is able to propagate the nucleic acid through several passages (e.g.. for at least 10 passages), and. preferably, the nucleic acid is stably integrated into the chromosomes of such cells. Thus, the cell line can propagate the nucleic acid for at least 20 passages, and more preferably significantly more than 20 passages (e.g.. at least about 25 passages, or even more). Regardless of the cell system, the ability to express a T-type calcium channel nucleic acid within host cells to produce an active channel permits the channel to be further studied. In this regard, the present invention provides a method of identifying a drug which affects T-type calcium channels. 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.. ⁴³Ca). 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. For example, the dye can be one such as those that fluoresce or change color in the presence of calcium, many of which are known to those of skill in the art (e.g.. Indo-1). Thereafter, the cells are exposed to a depolarizing solution containing high (e.g., about 50 mM) potassium concentration and a drug, and the reaction of the labile dye is compared to control cells. Using a labile dye affords the ability to assay many putative drugs quickly in a high throughput assay for putative drugs affecting T-type channels. For example, the initial screening can be carried out in 96 well plates. Moreover, dose-response data can be readily generated by exposing the cells to several concentrations of the same putative drug.

Once a putative drug is detected, its effect on the electrophysiology of the cell (e.g., single channel conductance, voltage dependency, activation kinetics, inactivation kinetics, and tail current of the cells) can be investigated in detail. Generally, the effect of the putative drug on T-type calcium currents is assessed by measuring the various electrophysiological parameters in the presence of various concentrations of the drugs and comparing the data to untreated (or sham-treated) control cells. Cells preferably are maintained in a continuous perfusion chamber during such experiments to facilitate changing solutions. The inventive method of identifying a drug which affects T-type calcium channels can employ any nucleic acid encoding a T-type calcium channel (or derivative thereof), such as those nucleic acids described herein. In fact, as several isoforms of T-type channel exist, the assay method can be repeated using nucleic acids encoding different isoforms to identify drugs that preferentially target a given isoform. or drugs which affect more than one isoform of T-type calcium channels.

Aside from affording an in vitro assay for detecting potential therapeutic or investigative drugs targeting T-type calcium channels, the method of expressing the T-type calcium channel nucleic acid can also be used in vivo. For example, as mentioned, several neurological and muscular diseases or disorders have implicated mutations affecting native nucleic acids encoding T-type calcium channels. The present invention, thus, provides a method of treating a disease or disorder associated with a deficiency in a native T-type calcium channel nucleic acid. The method involves introducing a vector having the T-type calcium channel nucleic acid into cells of a host in which native expression of the nucleic acid is deficient. Thus, for example, for treating cardiomyopathy associated with deficiencies in T-type calcium channels, the vector is introduced into myocardial cells. Similarly, for treating forms of epilepsy associated with deficiencies in T-type calcium channels, the vector is introduced into neurons (e.g.. thalamic neurons). Within the target cells, the nucleic acid within the vector is expressed to produce active T-type calcium channel. By similar methods, 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. Thus, the present invention is useful to treating disorders associated with over-expression of T-type calcium channels.

T-type channel proteins (such as whole T-type calcium channels, domains of such channels, chimeras including portions of T-type calcium channels, etc.) can be employed to generate antibodies (e.g.. immunoglobulins) to T-type calcium channels. Thus, the present invention provides an isolated and substantially purified antibody molecule recognizing an epitope on a T-type calcium channel. Such antibodies can be monoclonal antibodies or polyclonal antisera. Antibodies recognizing T-type calcium channels can be used to purify the channels from cell extracts or other solutions by standard methodologies (e.g., immunoprecipitation). Moreover, depending on the location of the epitopes for the antibodies on the T-type calcium channel, the antibodies can be used to affect the channel proteins present on the surface of cells. Thus, 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. Thus, for example, the antibodies can comprise polyclonal antisera obtained from innoculated animals. Alternatively, the antibody molecules can be monoclonal antibodies obtained from a cell line (e.g., a hybridoma cell line). Thus. 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.

Several examples are presented below to illustrate the invention. Taken together, the examples demonstrate the cloning of twelve novel proteins and their characterization as T-type calcium channel α subunits. These examples are included here for purely illustrative purposes; as such, they are not to be construed so as to limit the scope of any aspect of the invention.

Many procedures employed in the following examples are techniques routinely performed by one of ordinary skill in the art (see generally Sambrook et al.. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor. NY (1989)) and are not discussed in detail. However, some reagents and methods deserve specific description. Thus, for example, in vitro translation and expression were conducted as described previously (Schneider et al.. Receptors and Channels, 2, 255-70 (1995)). Xenopus laevis oocytes were prepared as described previously

(Bernal et al., J. Pharmacol. Exp. Ther., 282, 172-80 (1997)). To express proteins, 10 or 30 ng of capped cRNA was injected into the oocytes in a volume of 50 nl. For single channel recording, 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. 50 mM NaOH, 1 mM KOH, 0.1 mM EDTA, and 5 mM HEPES, adjusted to pH 7.4 with methanesulfonate. The osmolality of the 2 mM Ba²⁺ and 10 mM Ba²⁺ solutions was balanced by increasing the NaOH concentration as described (Lory et al., J. Physiol, (London), 429, 95-1 12 (1990)). Voltage and current electrodes (1.5-1.8 M tip resistance) were filled with 3 M KC1. Except as noted, data were acquired at 4 kHz using the pCLAMP system, and filtered at 1 kHz. Data were analyzed using pCLAMP software. Boltzman fits and linear regression were calculated using Prism.

EXAMPLE 1

This example demonstrates the cloning and characterization of putative T-type calcium channels.

A search of the Genbank library was conducted to identify clones identified as having some degree of homology to known calcium channel sequences. The search identified an expressed sequence tagged (EST) partial sequence in a human brain clone (H06096), which was used as a probe to screen a λgtlO cDNA library prepared from rat brain. Successive screening of the cDNA library identified five overlapping clones which were aligned to construct an entire cDNA sequence, termed αlG. The αl G cDNA was cloned into the pSP72™ vector and sequenced by standard computer-assisted sequencing. Using the αlG cDNA, the amino acid sequence of the αlG protein was deduced and compared to the sequences of other known calcium channel α subunits. By similar methods, homologous human (HI 9230 and R19524) and mouse (AA286626) EST clones were also identified and partially sequenced, and alternately spliced variants were identified. The deduced cDNA and amino acid sequences for eight full-length αlG T-type channels are set forth, respectively, as SEQ ID NOs: 1-8.

A second T-type calcium channel, termed αlH. was isolated by screening a human heart cDNA library with a fragment of the αlG sequence. An alternately spliced isoform was also identified. The full-length cDNA and amino acid sequences for these αlH T-type channels are set forth, respectively, as SEQ ID NOs:9 and 10. A third T-type calcium channel, termed all. was isolated by screening a rat brain cDNA library at low stringency using a fragment of the rat αlG gene. Fifty plaques were identified, many of which were not detected in a second screening. A third screening with a fragment from αlH identified two clones. Subsequent screening, and the use of the GenBank database, led to the identification of the full length rat and human cDNA and amino acid sequences, set forth at SEQ ID NOs: 1 1 and 12, respectively.

The αl G. αlH. and all amino acid sequences were compared to each other and a known calcium channel (αlE) to investigate the conservation of protein structure and function. The comparison indicates that the αlG, αlH. and all amino acid sequences within the putative membrane-spanning domains are about 90 % identical to each other, while the αlG, αlH. and all sequences are only roughly 40 % identical to the α IE clone.

Figures 1 A- IE indicate this conservation between the proteins. The conservation of charged residues, particularly in the S4 domains, is consistent with the role of the αlG, αlH, and all proteins as ion channels. However, two of the glutamates associated with ion specificity in other calcium channels have been replaced with aspartate. suggesting altered ion selectivity. Strikingly, αlG, αlH, and all display only low homology to sequences linking the membrane-spanning regions within each domain, and even less homology between the intracellular loops linking domains. Notably, neither αlG, αlH. nor all possesses sequences known to bind β subunits or Ca²⁺ ions. EXAMPLE 2

This example demonstrates the production of cell lines stably expressing the cloned αl G. αl H. and al l proteins.

HEK-293 cells were transfected with either the rat αlG cDNA (SEQ ID NO: l). the human αlH cDNA (SEQ ID NO:9). or the rat all cDNA (SEQ ID NO: l 1 ). As a control, cells were also transfected with human αlE plus human β3 (Schneider et al.. Receptors Channels, 2, 255-70 (1994); Murakami et al.. Eur. J. Biochem., 236, 138-43 (1996)). The DNA constructs included a neomycin resistance gene conferring resistance to G418. The cells were cultured under standard conditions using medium containing G418 to select for stable transformants.

Surviving clones were expanded and assayed for electrophysiological activity to determine the presence of channels within the membrane. Whole-cell currents were recorded from ruptured patches using an Axopatch 200 A amplifier. Digidata 1200 A/D converter, and pCLAMP 6.0 software. Data were digitized at 2 kHz and filtered at 1 kHz or off-line. All experiments were performed at room temperature. Pipettes were made out of TW- 150-6 capillary tubing (World Precision Instruments, Inc., Sarasota. FL), using a Model P-97 Flaming-Brown pipette puller (Sutter Instrument Co., Novato, CA). The internal pipette solution contained the following: 55 mM CsCl, 75 mM CsSO , 10 mM MgCl₂, 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 CaCl₂, 10 mM glucose, 5 mM HEPES, pH 7.4. The recording solution contained the following: 10 mM BaCl₂ solution (or 2 mM CaCl₂), 140 mM tetraethylammonium (TEA) chloride, 5 mM CsCl. 1 mM MgCl₂, 5 mM glucose, and 10 mM HEPES, pH adjusted to 7.4 with TEA-OH. Under these solution conditions the pipette resistance was typically 1.5-2.5 MΩ. Cell capacitance was measured by integrating the charging current during a 10 mV hyperpolarizing pulse (holding potential -80 mV).

Using these recording techniques, values for pA/pF were obtained for each cell line, which is a measure of current density normalizing for cell size. One clone (#N2) expressed the rat αlG protein and has a current density of 42 pA/pF. Another clone (#13), expressed the human αlH protein and exhibited a current density of 53 pA/pF. Three clones (#1 1, #19, and #25) expressed the rat all protein and exhibited current densities of 40 pA/pF, 45 pA/pF, and 55 pA/pF, respectively

EXAMPLE 3

This example demonstrates that the cloned putative T-type calcium channels exhibit T-type current-voltage relationships. Current traces were elicited by depolarizing voltage clamp pulses of the membranes of cells. The αlG. αlH. and all proteins were produced in Xenopus laevis oocytes by linearizing the DNA vectors containing the coding sequences, and transcribing the coding sequences in vitro by standard methods. Oocytes were then injected with the capped RNA.

Figures 2A-2E depict data obtained from these experiments using cells injected with αlG (Figure 2A). αlH (Figure 2B). and all (Figure 2C) and αlE (Figure 2D). These data indicate that cells expressing αlG. αlH. and al l exhibit T-type calcium current, while oocytes expressing αlE as well as uninjected oocytes (Figure 6A) do not.

Current voltage curves were developed using cells injected with αlG. αlH, all. and αlE. Figures 3A depicts such data generated in a 10 mM Ba²⁺ test solution. These data were transformed into conductance and fit with a Boltzman equation to determine the midpoint of activation (V_{0 5}). Gating potentials for αlG. αlH. and all (-38 ± 1 mV n=8, -44 mV ± 1 mV, n=10, and -31 mV ± 1 mV. n=6. respectively) were in accordance with the gating potential measured for the HEK-293 cells (-41 ± 1 mV, n=10), while αlE required significantly more positive potentials to open (-2.6 mV ± .4 mV. n=3).

To compare the characteristics with published values (Huguenard, Ann. Rev. Physiol, 58, 329-48 (1996)), the αlG current was recorded at varying concentrations of Ba²⁺. As indicated in Figure 3B, in solutions containing 2 mM Ba²⁺, V_{0 5} was -46.5 mV, and the slope factor (k) was 6.6 (n=7). However, when the Ba²⁺ concentration was 40 mM, V_{0 5} was recorded at -21 mV, presumably due to the results of barium on surface charge screening (see, e.g., Wilson et al.. J. Membrane Biol, 72, 1 17-30 (1983)). Similar values were recorded for αlH and all.

These results indicate that αlG. αlH, and all are low-voltage activated calcium channels (i.e., from about -60 mV to about -30 mV in 10 mM Ba²⁺).

EXAMPLE 4 This example demonstrates that the cloned putative T-type calcium channels exhibit T-type tail current.

Tail current was measured at -90 mV after first opening the channels with a voltage step to -10 mV. The voltage-dependence of tail current in cells expressing αlG (oocytes) αlH (HEK 293 cells), and all (HEK 293 cells) was measured at varying test potentials. As a control, tail current was also measured from a high voltage activated channel αlE, which Raw data from recordings data were fit with a single exponential and plotted as a function of depolarization potential (Figure 4). These results demonstrate that the tail currents for the cloned αl G. αlH. and all calcium channels are voltage-dependent, consistent with known T-type calcium tail currents. Additionally, these data demonstrate that the tail current for each of the cloned channels is between about 1 ms and about 10 ms following repolarization to a membrane potential from about -80 mV to about -60 mV in a solution with a barium concentration of from about 10 mM to about 40 mM.

EXAMPLE 5

This example demonstrates that the cloned putative T-type calcium channels exhibit T-type single channel conductance.

Measurement of 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 BaCl₂, 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)). Single channel amplitudes were measured by averaging the values obtained from Gaussian fits to all-points histograms of traces with openings, selected openings, or amplitude histograms of idealized openings. It has been reported that some oocytes contain a native 9 pS channel. These endogenous channels can be distinguished by their 2-fold larger current amplitudes at the potentials tested (e.g., -20 mV, / = 0.8 for endogenous channels as opposed to 0.4 pA for αlG). However, such endogenous channels were not detected either at the whole cell or single channel level in the oocytes tested.

Current through the main open state of each open channel was measured at each potential and plotted against each test potential. Single channel currents for several patches were then averaged and plotted as a function of test potential, wherein the slope of the plot indicated the single channel conductance. The average slope conductance of the αlG channel was measured at 7.5 ± 1.5 pS, which corresponds with the reported values for T-type calcium channels (Hugenard, Ann. Rev. Phsysiol, 58, 329-48 (1996)). Similar results were also obtained with both αlH (10.8 ± 1.4 pS). Data collected from recordings of the all channels indicate that they open to two distinct amplitudes, The conductance for the small amplitude all openings was measured at 3.9 ± 0.5 pS, while that for the large all openings was measured at 1 1.4 ± 0.5 pS).

These results indicate that the cloned αlG, αlH, and all proteins exhibit T-type single-channel conductance (e.g.. from about 4 to about 12 pS). EXAMPLE 6

This example demonstrates that a cloned T-type calcium channel can be used for identifying a drug which affects T-type calcium channels. HEK-293 cells were subjected to treatment as indicated above in Example 3, except that an experimental group of cells were exposed to a solution containing 1 μM mibefradil. a known inhibitor of T-type calcium current. As depicted in Figure 5 A, the presence of mibefradil almost completely abolished T-type current in cells expressing αlG. Cells expressing either αlG or αlH were similarly treated using various concentrations of mibefradil to determine a dose-response relationship. These results, depicted in Figure 5B. demonstrate that about 50% inhibition was achieved at a mibefradil concentration of 1 μM.

All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

What is claimed is:

1. A isolated or substantially purified nucleic acid encoding a protein comprising at least one domain of a T-type calcium channel ╬▒ subunit.

2. The nucleic acid of claim 1. wherein said protein comprises an entire T- type calcium channel ╬▒ subunit.

3. The nucleic acid of claim 2, wherein said protein comprises SEQ ID NO: 13.

4. The nucleic acid of any of claims 1-3, wherein said calcium channel begins to gate from about -60 mV to about -30 mV in 2 mM Ba²⁺.

5. The nucleic acid of any of claims 1-4, wherein said calcium channel exhibits a tail current of from about 1 ms to about 10 ms following repolarization to a membrane potential from about -80 mV to about -60 mV in a solution with a barium concentration of from about 10 mM to about 40 mM.

6. The nucleic acid of any of claims 1-5, wherein said calcium channel exhibits a single channel conductance of from about 4 pS to about 1 1 pS in a solution with a barium ion concentration of about 100 mM.

7. An isolated or substantially purified nucleic acid hybridizing to the nucleic acid of any of claims 1-6.

8. An isolated or substantially purified nucleic acid hybridizing to the nucleic acid of claim 7.

9. The nucleic acid of claim 8 comprising a sequence encoding at least one domain of a T-type calcium channel ╬▒ subunit.

10. A vector comprising the nucleic acid of any of claims 1-9.

1 1. A cell into which the vector of claim 10 has been introduced.

12. The cell of claim 1 1, which expresses said nucleic acid to produce said protein.

13. The cell of claim 1 1 or 12, which stably expresses said nucleic acid to produce said protein.

14. A population of cells consisting essentially of cells according to any of claims 1 1-13.

15. An established cell line consisting essentially of cells according to any of claims 1 1-13.

16. A method of identifying a drug which affects T-type calcium channels, said method comprising expressing a T-type calcium channel in a cell, exposing said cell to a putative drug, and measuring the calcium flux through the membrane of said cell in response to a change in membrane potential.

17. The method of claim 16. wherein said calcium flux is assayed by using a calcium-sensitive labile dye within said cell.

18. The method of claim 16. wherein said calcium flux is assayed by measuring the electrophysiological properties of said cell.

19. The method of claim 16. wherein said calcium channel comprises SEQ ID

NO: 13.

20. An isolated or substantially purified immunoglobulin recognizing an epitope on a T-type calcium channel protein.

21. A cell in vitro which produces the immunoglobulin of claim 20. 22. An established cell line consisting essentially of cells according to claim

21.