WO1993008469A1

WO1993008469A1 - Recombinant calcium channel polypeptides

Info

Publication number: WO1993008469A1
Application number: PCT/US1992/009109
Authority: WO
Inventors: Stanley C. Froehner; Elizabeth L. R. Barry
Original assignee: Trustees Of Dartmouth College
Priority date: 1991-10-23
Filing date: 1992-10-21
Publication date: 1993-04-29

Abstract

Disclosed is an essentially pure, recombinant, immunoreactive alpha-1 polypeptide subunit, or a portion thereof, of the human voltage-sensitive calcium channel protein expressed on human small cell lung carcinoma. Also disclosed is an isolated nucleic acid encoding this polypeptide and an expression vector containing such nucleic acid. In addition, methods are provided using this polypeptide for the detection of antibodies against calcium channel protein, and diagnosis of small cell lung carcinoma in a human subject, and for the detection and treatment of Lambert-Eaton Syndrome.

Description

RECOMBINANT CALCIUM CHANNEL POLYPEPTIDES

BACKGROUND OF THE INVENTION

Small cell (or "oat cell") carcinoma of the lung (SCCL) is a highly malignant tumor of epithelial cell origin, the development of which is often linked to cigarette smoking. These carcinoma are the most aggressive of lung tumors: they metastasize widely, and are essentially incurable by surgical means (see Robbins e_t al. -■ Pathologic Basis of Disease (3rd Ed.) 1984 by the W.B. Saunders Co., Philadelphia, PA, p. 753).

Because of the aggressive nature of this carcinoma, its early diagnosis is a prerequisite to successful treatment. This has been accomplished by screening individuals at high risk, particularly males over 40 years of age who smoke 40 or more cigarettes per day, for SCCL by culturing sputum samples and/or taking chest radiographs (Minna, in Harrison's Principles of Internal Medicine. 11th Edition (1987) (Braunwald et. al.. eds.) McGraw-Hill Book Co., New York pp. 1116-1118). However, when the carcinoma is capable of being detected by these methods, it most likely has progressed too far to be effectively treated; the life expectancy of the an afflicted person is only about two years. SCCL cells exhibit many characteristics of neurons. They, like central and peripheral cell neurons, as well as cardiac, skeletal, and smooth muscle cells, express high levels of the voltage- dependent calcium channel protein. This marker is a membrane-spanning, multisubunit protein that allows for the controlled entry of calcium ions into cells from the extracellular environment. The opening of the channel to allow influx of calcium ions requires a depolarization to a certain level of the potential difference between the inside of the cell and the exteral medium, the rate of influx depending on this voltage difference. The presence of SCCL may be detected in tissue specimens with antibodies specific for this protein (U.S. Patent No. 4,954,436). Patients with SCCL have a high incidence of

Lambert-Eaton Syndrome (LES), a neuromuscular disease characterized by deficient neurotransmitter release from the neuromuscular presynaptic terminal (Lambert et al. (1982) Muscle Nerve 5:529-535). This condition appears to be the result of the action of anti-calcium channel autoantibodies developed in response to the presence of calcium channel protein on SCCL cells. In some cases, the appearance of LES precedes the diagnosis of the accompanying SCCL, suggesting that these calcium channel antibodies may be present before the tumor can be detected.

SUMMARY OF THE INVENTION

This invention pertains to an essentially pure recombinant alpha-1 polypeptide subunit, or portion thereof, of the human voltage-dependent calcium channel on small cell lung carcinoma. An "essentially pure polypeptide" refers to a polypeptide that is substantially free of other peptide components such that it is considered homogeneous by SDS-PAGE, and/or can be unambiguously sequenced. In preferred aspects of the invention, this recombinant polypeptide, or portion thereof, is immunoreactive, and may have, as at least a portion of its amino acid sequence, the sequences set forth in the Sequence Listing as SEQ ID NO:l, 2, or 3. The recombinant polypeptide of the invention may also be in the form of a fusion protein. This invention also provides an isolated nucleic acid, such as DNA or expression vector, having a nucleotide sequence encoding the SCCL alpha-1 calcium channel polypeptide, or portion thereof. In some embodiments of the invention, the nucleic acid is a DNA having the sequences listed in the Sequence Listing as SEQ ID NOS:4, 5, and 6.

The recombinant polypeptides of the invention can be used as a part of an immunochemical assay for detecting autoantibodies to the human voltage- dependent calcium channel, and for diagnosing SCCL, neuroblastoma, and other tumors which express a surface protein recognized by antibodies specific for the voltage-sensitive calcium channel.

A preferred assay is a solid phase immunometric assay. In this method, an immunoabsorbent is provided which includes a solid phase to which is attached the polypeptide of the invention. The immunoabsorbent is incubated with a sample of a biological fluid (e.g., blood, serum, ascites, or plasma) taken from the subject, under conditions conducive for the binding of an anti-human calcium channel antibody to the immunoabsorbent-bound polypeptide. The binding of the antibody to the immunoabsorbent is determined after separation of the immunoabsorbent from the sample. The presence of the bound antibody is indicative of the presence of autoantibodies against the voltage- dependent calcium channel, and hence of SCCL in the subject. The polypeptides of the invention can also be a part of a kit useful for detecting SCCL or Lambert-Eaton Syndrome. This kit includes the solid-phase-bound polypeptide with which a sample from a potentially afflicted subject is contacted, and a labelled anti-human antibody, or a human antibody- binding portion thereof, used to detect the presence of human anti-calcium channel antibodies.

The recombinant polypeptides of the invention can also be used as a part of a method for detecting, diagnosing, and treating Lambert-Eaton Syndrome. In this method, an immunoreactive, recombinant polypeptide of the invention, or immunoreactive portion thereof, is provided and administered to a subject in an amount sufficient to block the binding of anti-calcium channel protein antibody to calcium channel protein on neurotransmitter-producing cells in the subject.

BRIEF DESCRIPTION OF THE FIGURES The foregoing and other objects of this invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which: FIG. 1 is a schematic representation of the primers used for PCR, indicating the positions of the residues corresponding to the numbering in the skeletal muscle calcium channel and their locations in the putative domain structure of the channel; and FIG. 2 is a schematic representation of the overlapping products of PCR using (a) primers 1 (SEQ ID NO:7) and 3 (SEQ ID NO:9), (b) primers 1 (SEQ ID NO:7) and 4 (SEQ ID NO:10), (c) primers 2 (SEQ ID NO:8) and 3 (SEQ ID NO:9), and (d) primers 2 (SEQ ID NO:8) and 4 (SEQ ID NO:10).

DESCRIPTION OF THE INVENTION

It has been discovered that recombinant forms of the voltage-dependent calcium channel expressed on human SCCL cells have a nucleic acid sequence and corresponding amino acid sequence different than those of other human and animal forms of this subunit. This invention pertains to these essentially pure, recombinant polypeptide subunits, or portions thereof. The human voltage-dependent calcium channel protein consist of two large subunits, alpha-1 and alpha-2, having molecular weights of about 170 - 210 kilodaltons (kD) , and 150 - 170 kD, respectively, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Different forms of this protein are found on human and animal noncancerous excitable cells as well as on various human and animal noncancerous muscle cells. The cDNA and corresponding amino acid sequence of various human and animal muscle and brain forms of the calcium channel protein have been reported. See, for example, Koch et al. (1990) J. Biol. Chem. 21^:17786-17791; PCT/US89/01408; and Tanabe et al.. (1987) Nature 3_28:313-318. In one of its aspects, the invention is a DNA which codes for recombinant forms of the alρha-1 subunit found on human SCCL. Partial nucleic acid sequences and the corresponding amino acid sequences of the alpha-1 subunit of the voltage-dependent calcium channel expressed on human SCCL cells are shown in the Sequence Listing as SEQ ID NOS:l-3 and NOS:4-6, respectively. These partial sequences can be used as probes to isolate the entire gene sequence for the SCCL alpha-1 subunit. In addition, the partial and complete sequences may also be used to produce recombinant forms of the alpha-1 subunit but standard methodologies, including ligating the sequence into an expression vector, transforming an appropriate prokaryotic or eucaryotic host cell, culturing the cell to produce the polypeptide, possibly as a fusion protein, and then purifying the subunit from the cells.

The DNA encoding the recombinant SCCL alpha-1 polypeptides can be prepared as follows. Total cellular RNA is isolated from SCCL cells according to standard methods such as those described in Sambrook et al. (Molecular Cloning, A laboratory Manual,- 2nd Ed., Cold Spring Harbor Laboratory Press (1989) pp. 7.6-7.35). Poly A+ RNA is isolated and reversed transcribed and amplified by the polymerase chain reaction. Using the sequences available for other alpha-1 calcium channel polypeptides, such as the rabbit skeletal and cardiac muscle calcium channels, primers have been assigned that correspond to highly conserved regions of the alpha-1 subunit that are only poorly conserved with sodium channel sequences. These are depicted in FIG. 1 and in the Sequence Listing, SEQ ID NOS:7-10. The polymerase chain reaction (PCR) was then performed on first strand cDNA derived from poly A⁺ RNA isolated from SCCL cell lines. The PCR products were then subcloned and sequenced by, for example, the dideoxynucleotide chain termination procedure. The methodology used to isolate cDNA clones and to generate the cDNA fragments here claimed is described in the EXAMPLES set forth below.

Alternatively, the cDNA reversed transcribed from the SCCL RNA can be cloned directly into and amplified in a prokaryotic or eucaryotic host for amplification. Expression systems such as expression vectors and host cells transformed with this alpha-1 calcium channel subunit-encoding DNA are also provided by the invention. In another of its aspects, this invention is an essentially pure, recombinant form of the SCCL alpha-1 subunit. An "essentially pure polypeptide" refers to a polypeptide that is substantially free of other peptide components such that it is considered homogeneous by SDS-PAGE, and/or can be unambiguosly sequenced. This polypeptide may be apart of a fusion protein containing, for example, a signal sequence or other peptide sequences.

The recombinant alpha-1 subunit of the voltage-dependent calcium channel can be used in immunochemical methods of detecting and diagnosising SCCL in a human subject. Such immunochemical assays employing the polypeptides can take a variety of forms. The preferred type is a solid phase immunometric assay. In assays of this type, the purified, recombinant SCCL alpha-1 polypeptide is immobilized on a solid phase to form an immunoabsorbent. The immunoabsorbent is incubated with the biological sample to be tested, such as blood, serum, plasma, or any body fluid likely to have antibodies in it. The incubation is performed under consitions suitable for the formation of an antigen-antibody complex between an anti-human calcium channel antibody in the fluid and an immunoabsorbent- bound polypeptide. The immunoabsorbent is then separated from the sample, and a labeled anti-(human IgG) antibody is used to detect human anti-calcium channel antibody bound to the immunoabsorbent. The amount of label associated with the immunoabsorbent is compared to positive and negative controls to assess the presence or absence of anti-calcium channel antibody, the presence of the bound antibody being indicative of the presence of SCCL in the subject.

The immunoabsorbent can be prepared by adsorbing or coupling purified polypeptide to a solid phase. Various solid phases can be used, such as beads formed of glass, polystyrene, polypropylene dextran, or other material. Other suitable solid phases include tubes or immunotitre plates formed from or coated with these materials.

The recombinant polypeptides can be either covalently or non-covalently bound to the solid phase. For example, the polypeptides can be covalently bonded to the solid phase using techniques involving amide or ester linkage or adsorption. After the recombinant calcium channel polypeptide is affixed to the solid phase, the solid phase can be post-coated with an animal protein, e.g., 3% fish gelatin or bovine serum albumin. This provides a blocking protein which reduces nonspecific adsorption of protein to the immunoabsorbent surface. The immunoabsorbent functions to insolubilize anti-calcium channel antibody in the liquid sample tested. In blood screening for anti-calcium channel antibody, the immunoabsorbent is incubated with blood plasma or serum. Before incubation, plasma or serum is diluted with normal animal plasma or serum. The diluent plasma or serum is derived from the same animal species that is the source of the anti-(human IgG) antibody. The preferred anti-(human IgG) antibody is goat anti-(human IgG) antibody. Thus, in the preferred format, the diluent would be goat serum or plasma. The optical dilution factor for human plasma and serum is about 10-11 fold.

The conditions of incubation, e.g. pH and temperature, and the duration of incubation can be optimized by routine experimentation. Generally, the incubation will be run for 1-2 hours at about 45°C, in a buffer having a pH of about 7 - 8.

After incubation, the immunoabsorbent and the sample are separated. Separation can be accomplished by any conventional technique such as sedimentation or centrifugation. The immunoabsorbent then is washed free of sample to eliminate any interfering substances. To assess human antibody bound to the immunoabsorbent, the immunoabsorbent is incubated with the labelled anti-(human IgG) antibody (tracer). Generally, the immunoabsorbent is incubated with a solution of the labeled anti-(human IgG) antibody which contains a small amount (about 1%) of the serum or plasma of the animal species which serves as the source of the anti-(human IgG) antibody. Anti-(human IgG) antibody can be obtained from any animal source. However, goat or rabbit anti-(human IgG) antibody is preferred. The anti-(human IgG) antibody can be an antibody against the Fc fragment of human IgG, for example, goat anti-(human IgG) Fc antibody.

The anti-(human IgG) antibody or anti-(human IgG)Fc can be labelled with a radioactive material such as ¹²⁵Iodine; labeled with an optical label, such as a fluorescent material; or labeled with an enzyme such as horseradish peroxidase. The anti-human antibody can also be biotinylated and labeled avidin used to detect its binding to the immunoabsorbent.

After incubation with the labeled antibody, the immunoabsorbent is separated from the solution and the label associated with the immunoabsorbent is evaluated. Depending upon the choice of label, the evaluation can be done in a variety of ways. The label may be detected by a gamma counter if the label is a radioactive gamma emitter. Alternatively, the label may be detected by a fluorimeter, if the label is a fluorescent material. In .the case of an enzyme label detection may be done colorimetrically employing a substrate for the enzyme.

The amount of label associated with the immunoabsorbent is compared with positive and negative controls in order to determine the presence of anti-calcium channel antibody. The controls are generally run concomitantly with the sample to be tested. A positive control is a serum containing antibody against calcium channel; a negative control is a serum from healthy individuals which do not contain antibody against calcium channel protein. For convenience and standardization, reagents for the performance of the immunometric assay can be assembled in assay kits. A kit for screening blood, for example, can include: (a) an immunoabsorbent such as, e.g., a polystyrene bead coated with a recombinant alpha-1 subunit of the calcium channel protein;

(b) a diluent for the serum or plasma sample such as, e.g., normal goat serum or plasma; (c) an anti-(human IgG) antibody such as, e.g., goat anti-(human IgG) antibody in buffered, aqueous solution containing about 1% goat serum or plasma; (d) a positive control, i.e., serum containing antibody against the calcium channel protein; and (e) a negative control, such as, e.g., serum from healthy individuals which does not contain antibody against the calcium channel protein.

If the label is an enzyme, an additional element of the kit can be the substrate for the enzyme. Another type of assay for anti-calcium channel antibody is an antigen sandwich assay. In this' assay, a labelled recombinant calcium channel polypeptide of this invention is used in place of anti-(human IgG) antibody to detect anti-calcium channel antibody bound to the immunoabsorbent. The assay is based in principle on the bivalency of antibody molecules. One binding site of the antibody binds the antigen affixed to the solid phase; the second is available for binding the labeled antigen. The assay procedure is essentially the same as described for the immunometric assay except that after incubation with the sample, the immunoabsorbent is incubated with a solution of labeled core polypeptide. The calcium channel polypeptide can be labeled with radioisotope, an enzyme, etc. for this type of assay.

In a third format, the bacterial protein. Protein A, which binds the Fc segment of an IgG molecule without interfering with the antigen antibody interaction can be used as the labeled tracer to detect anti-calcium channel antibody adsorbed to the immunoabsorbent. Protein A can be readily labeled with a radioisotope, enzyme, or other detectable species.

An additional use of the recombinant alpha-1 polypeptide is in the treatment of Lambert-Eaton syndrome. The polypeptide is administered to an afflicted subject, with a pharmaceutically aceptible carrier, in an amount sufficient to block the binding of anti-calcium channel protein antibody to calcium channel protein on neurotransmitter producing cells in the subject. Effective dosages of the recombinant. polypeptide and modes of its administration in the treatment of LES can be determined by routine experimentation. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacterial and fungi. The carrier can be a solvent or dispersion medium. The prevention of the action of microorganisms can be brought about by various antibacterial and^" antifungal agents. Prolonged absorption of the injectable therapeutic agents can be brought about by the use in the compositions of agents delaying absorption.

Sterile injectable solutions are prepared by incorporating the recombinant polypeptide in the required amount in the appropriate solvent, followed by filtered sterilization.

The recombinant polypeptide may be administered parenterally or intraperitoneally. Solutions of the therapeutic agent as pharmacologically acceptable salts can be prepared in water or some other physiologically acceptable solvent.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.

The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

Supplementary active ingredients can also be incorporated into the compositions.

The invention is illustrated further by the following non-limiting examples:

EXAMPLES

1. Preparation of mRNA

The method of Bradley e_t al. (Biotechiques (1988) 6_.:114-116) was used to purify poly A⁺ RNA from tissue culture cells. H146 SCCL cells (Gazdar (1980) Cancer Research 4_0ϊ3502-3507 and Kim (1989) Soc. Neurosci. Abstr. .15.:824) were pelleted by centrifugation and homogenized in the presence of 2% SDS. After incubation with proteinase K, the cell lysate was batch-absorbed to oligo [dT] cellulose, and the poly A⁺ RNA was eluted in buffer without salt. The yield was approximately 60 μg of poly A⁺RNA from one ml of packed cells.

RNA analyzed on a formaldehyde/ agarose gel migrated as a broad smear from much greater than 9.5 kilobases (kb) to about 0.25 kb. In addition, several discrete bands of RNA were seen within the smear. The RNA was analyzed by Northern blotting (see, e.g., Goldberg (1980) Proc. Natl. Acad. Sci. (U.S.A.) 5794) and hybridized to a radioactive probe for actin. A single sharp band was obtained, indicating that the RNA was undegraded. This RNA was used for the determination of SCCL calcium channel sequences using the Polymerase Chain Reaction (PCR) as described below. The same procedure was also used for the purification of RNA from a mouse muscle cell line (C2) which were induced to form myotubes in culture (Silberstein (1981) Nature 295:143-145). This RNA was used as a control in the PCR reaction since it is expected to express a muscle calcium channel closely related to the rabbit muscle channel. 2. Design of Primers for PCR

Each PCR reaction requires a pair of primers which flank the region of DNA to be amplified and hybridize to opposite strands. Four primers were designed, which could be used in four combinations for PCR reactions. Their sequences were based on regions which are perfectly or almost perfectly conserved between the rabbit skeletal muscle and cardiac calcium channels (94 to 100% idential with any differences near the 5' end of the primer). The sequence homology between calcium and sodium channels in these regions ranges from approximately 50% to 60% (Tanabe (1987) Nature 228:313-318 and Trimmer (1989) Neuron 3.:33-49). Thus, these primers are specific for calcium channels relative to other types of cation channels. The position of the residues indicated in FIG. 1 below correspond to the numbering in the skeletal muscle calcium channel (Tanabe (1987) Nature 328:313-318) . Their location of these primers in the putative domain structure of the channel is noted in parenthesese.

One of two types of restriction sites (underscored) were included near the 5* ends of the primers to facilitate directional subclong: ECP Rl sites for the two primers hybridizing to the sense strand; and BAM HI sites for the two primers hybridizing to the antisense strand. In addition, four nucleotides were included 5' to the restriction sties to insure that cutting could occur at these sites. Other features which were taken into consideration in the design of these primers were the G + C composition (close to 50% to maximize specificity) , and complementarity of primer pairs which was minimized to avoid formation of primer dimers. These primers were synthesized locally, and purified on Nensorb prep columns (Johnson (1990) Biotechniques 8:424-429).

3. Polymerase Chain Reaction

Poly A⁺RNA, purified from cultured cells as described above, was reverse transcribed in order to make cDNA templates for PCR Current Protocols in Molecular Bioloσv, (1989) Ausubel et al. (eds.)), John Wiley and Sons, New York, pp. 5.5.2-5.5.4). Random hexamers were used to prime the reverese transcriptase reaction. The cDNAs were purified by phenol extraction and aliquots were used for PCR reactions. PCR was performed on an automated instrument

(Perkin-Elmer Cetus) using the following program: 1) 94°C for 3 min; 2) 30 cycles of: 94°C for 1 min for denaturation; 65°C for 1 min for primer annealing; 72°C for 2 min + 2 sec/cycle for primer extension; 3) 72°C for 7 min; 4) 4°C. The conditions for the PCR reaction were 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1.5 mM MgCl2 0.001% gelatin, 200 μM of each dNTP, 0.2 μM of each primer, and 2.5 units of DNA polymerase (amplitaq, Perkin-Elmer) per 100 μl reaction. In addition, the reaction contained template cDNA synthesized from 1 μg of poly A⁺ RNA.

Positive and negative controls were included in each PCR run. A clone (1 ng) containing the entire rabbit skeletal muscle calcium channel cDNA (Ellis (1988) Science 141:1661-1664) was used as template for a positive control. In another type of positive control (for the reverse transcription reaction) cDNA synthesized from poly A+RNA isolated from C2 cells was used as template since this muscle cell line is expected to express a calcium channel with high homology to the rabbit skeletal muscle channel. Two negative controls were also used: in one no template DNA was included in the reaction mixture, and in the other the template was the products isolated from a mock reverse transcriptase reaction in which only the enzyme was left out. The products of the PCR reactions were analyzed by electrophoresis on 3% Nuseive, 1% Seakem agarose gels in order to visualize small DNA products.

All four combinations of primers gave products of the expected size when the cloned calcium channel cDNA was used as template: primers 1 (SEQ ID NO:l) and 3 (SEQ ID NO:3) gave a 934 base pair product; primers 1 (SEQ ID NO:l) and 4 (SEQ ID NO:4) gave a 1275 base pair product; primers 2 (SEQ ID NO:2) and 3 (SEQ ID NO:3) gave a 390 base pair product; and primers 2 (SEQ ID NO:2) and 4 (SEQ ID NO:4) gave a 731 base pair product. These represent overlapping products as diagramed in FIG. 2.

No products were seen when the reaction was run in the absence of any template DNA. When PCR was performed on the reverse transcribed cDNA from C2 and H146 SCCL cell lines, the products described in TABLE 1 below were seen as bands on agarose gels.

TABLE 1

primers 1 (SEQ ID NO:l) and 3 (SEQ ID NO:3) :

1) C2- a doublet around 934 bp (equal amounts of both bands)

2) H146- a doublet around 934 bp (more of the lower band in the doublet)

primers 1 (SEQ ID NO:l) and 4 (SEQ ID NO:4):

1) C2- a doublet around 1275 bp (equal amounts of both bands)

2) H146- a doublet around 1275 bp (more of the lower band in the doublet)

primers 2 (SEQ ID NO: . and 3 (SEQ ID NO:3):

1) C2- a single band at around 390 bp

2) H146- a doublet at around 390 bp and another band at approximately 250 bp

primers 2 (SEQ ID NO:2) and 4 (SEQ ID N0:4):

1) C2- a single band at around 731 bp

2) H146- a single band at around 731 bp

The synthesis of multiple products in the PCR reactions suggests that both of the cell lines express more than one type of calcium channel. These different forms of calcium channels may be generated either from multiple genes or by alternative splicing of a single gene. It has been suggested from the cloning of aortic calcium channels that alternative splicing is a mechanism for generating channel diversity (Koch (1990) J. Biol. Chem. 265:17786- 17791) . 4. Subcloning and Sequencing of PCR Products

The PCR products from two of the sets of primer pairs (1 (SEQ ID NO:l) and 3 (SEQ ID NO:3) and 2 (SEQ ID NO:2) and 3 (SEQ ID NO:3)) were subcloned and sequenced. This region corresponds to the entire fourth domain of the calcium channel. For subcloning, the PCR products were purified by phenol extraction and ethanol precipitation and digested with ECO Rl and Bam HI. The products of the restriction digest were then isolated as a band from a low melt

Nusieve/agarose gel. The Bluescript vector (Stratagene) was also digested with ECO Rl and BAM HI and isolated on a low melt agarose gel. The vector and PCR product were ligated and used to transform XL-1 Blue bacteria. The sequences of the recombinant clones were determined by the dideoxynucleotide chain termination procedure (Sanger e al. (1971) Proc. Natl. Acad. Sci. (USA) 74.:5463-5467) . Initially, the SK and KS Bluescript primers were used to sequence in from the ends of the inserts.

Subsequently, internal primers were designed based on the partial sequences obtained and used to complete the sequencing. The sequences were compared to those of the skeletal and cardiac muscle calcium channels using the Intellegenetics On-line software. Three types of putative calcium channels were found. Two types (SCCL-B, (SEQ ID NO:6), SCCL-C, (SEQ ID NO:7)) were highly homologous to the skeletal muscle channel and to each other, while the third (SCCL-A, (SEQ ID NO:5)) was significantly less homologous, as discussed below. In the region sequenced, between primers 1 (SEQ ID NO:l) and 3 (SEQ ID NO:3) (i.e., between domains IIIS6 and IVS6), the degree of homology between the cardiac and skeletal muscle calcium channes is approximately 79% and 74% at the nucleotide and amino acid levels respectively, if gaps are not considered. There are two gaps in the sequence comparison in this region: (1) a large stretch of 8 amino acids located between domains IVS3 and IVS4 is present in the skeletal muscle channel but not the cardiac channel; (2) 2 amino acids which are located between domains IVS5 and IVS6 are found in the cardiac but not the skeletal channel.

The SCCL-B (SEQ ID NO:6) and SCCL-C (SEQ ID NO:7) clones are.highly homologous to each other and to the skeletal muscle calcium channel. The homology between the two SCCL clones is 90% (nucleotide) and 95% (amino acid) . Both of these clones are approximately 90% (nucleotide) and 95% (amino acid) homologous to the skeletal muscle calcium channel in the region betweenprimers 1 and 3. The degree of homology to the cardiac channel is considerably less: 79% (nucleotide) and 75% (amino acid) . These comparisons do not take gaps in the sequence comparison into consideration. The gap between domains IVS3 and IVS4 (see above) is larger: a stretch of 19 amino acids is missing from the SCCL clones but present in the skeletal channel. Compared to the cardiac sequence, the SCCL clones are missing only 11 amino acids in this region since the cardiac is already 8 short of the skeletal channel. The 2 amino acids between IVS5 and IVS6 unique to the cardiac channel are also not found in the SCCL clones. Finally, SCCL-B (SEQ ID NO:9) is missing a single isolated amino acid which is found in all the other types of calcium channels. Thus, while the SCCL clones are highly homologous to the skeletal channel, they appear to have features unique to SCCL. Furthermore, it is expected that the region sequenced here will be one of the most conserved in the channels. The amino and carboxy- terminal portions of the channels may be much less strongly conserved, as is seen in a comparison of the cardiac and skeletal muscle channels.

The third type of SCCL clone (SCCL-A) was significantly less homologous to cardiac and skeletal muscle calcium channels than were SCCL-B and SCCL-C. It was found as a product of only one primer pair (2 (SEQ ID NO:2) and 3 (SEQ ID NO:3)) but not the other (1 (SEQ ID NO:l and 3 (SEQ ID NO:3)), and therefore only the sequence in this smaller region could be compared. In addition, primer 2 (SEQ ID NO:2) was able to prime at a second site giving rise to both the 250 bp product and the larger product of the doublet around 390 basepairs seen when PCR was performed on SCCL cDNA. The degreee of homology to the skeletal channel was 70% (nucleotide) and 60% (amino acid). However, there are two gaps in the sequence comparison in this region: (1) 8 amino acids which are found only in the SCCL-A clone; and (2) 3 amino acids which are absent in the SCCL-A clone. The SCCL clone (SCCL-A) has sequence homology with a more recently characterized rabbit brain calcium channel (Mori et al. (1991) Nature 3^^:398-402). The nucleic acid sequence identity is 89.7%, and the amino acid sequence identity is 98.2%. Therefore, this clone is thought to represent a human neuronal calcium channel. EOUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Froehner, Stanley C.

Barry, Elizabeth L.R. (ii) TITLE OF INVENTION: DNA Encoding Human

Calcium Channels (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Lahive & Coc field

(B) STREET: 60 State Street, Suite 510

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02109

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version

#1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/781,421

(B) FILING DATE: 23-OCTOBER-1991

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: DeConti, Giulio A. Jr.

(B) REGISTRATION NUMBER: 31,503

(C) REFERENCE/DOCKET NUMBER: DCI-029PC (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-227-7400

(B) TELEFAX: 617-227-5941 (2) INFORMATION FOR SEQ ID NOil:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Oryctolagus cuniculus (F) TISSUE TYPE: Skeletal muscle

(ix) FEATUREt

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..30

(D) OTHER INFORMATION: product= "nucleotide primer" (x) PUBLICATION INFORMATION: (A) AUTHORS: Tanabe,

(C) JOURNAL: Nature

(D) VOLUME: 328

(F) PAGES: 313-318

(G) DATE: 1987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CAAGGAATTC GAGCTGGACA AGAACCAGCG 30

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Oryctolagus cuniculus

(F) TISSUE TYPE: skeletal muscle (ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..30

(C) JOURNAL: Nature

(D) VOLUME: 328

(F) PAGES: 313-318

(G) DATE: 1987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GTGGGAATCC ATCAAGTCCT TCCAGGCCCT 30

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE:

(A) ORGANISM: Oryctolagus cuniculus (F) TISSUE TYPE: skeletal muscle (ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..30

(C) JOURNAL: Nature

(D) VOLUME: 328

(F) PAGES: 313-318

(G) DATE: 1987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CAGGGGATCC AAGTTGTCCA TGATGACAGC 30

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..30

(D) OTHER INFORMATION: /product= "nucleotide primer" (x) PUBLICATION INFORMATION: (A) AUTHORS: Tanabe,

(C) JOURNAL: Nature

(D) VOLUME: 328

(F) PAGES: 313-318

(G) DATE: 1987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GTTCGGATCC AGATCTTCTT GATGATGGCC 30

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 359 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(F) TISSUE TYPE: Small Cell Lung Carcinoma

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CGCAAACTCC ATAAGCTTAT GTCTGTCTGC TGATCGCCAT GCTCTTCTTC ATCTATGCCA 60

TCATTGGGAT GCAGGTGTTT GGTAACATTG GCATCGACGT GGAGGACGAG GACAGTGATG 120

AAGATGAGTT CCAAATCACT GAGCACAATA ACTTCCGGAC CTTCTTCCAG GCCCTCATGC 180

TTCTCTTCCG GAGTGCCACC GGGGAAGCTT GGCACAACAT CATGCTTTCC TGCCTCAGCG 240

GGAAACCGTG TGATAAGAAC TCTGGCATCC TGACTCGAGA GTGTGGCAAT GAATTTGCTT 300

ATTTTTACTT TGTTTCCTTC ATCTTCCTCT GCTCGTTTCT GATGCTGAAT CTCTTTGTC 359 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 822 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (F) TISSUE TYPE: small cell lung carcinoma (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CGCAAACTCC ATAACCAATG TGTACAGTAT GCCCTGAAGG CCCGCCCACT GAGGTGCTAC 60

ATTCCCAAAA ACCCATACCA GTACCAGGTG TGGTACATTG TCACCTCCTC CTACTTTGAA 120

TACCTGATGT TTGCCCTCAT CATGCTCAAC ACCATCTGCC TCGGCATGCA GCACTACAAC 180

CAGTCGGAGC AGATGAACCA CATCTCAGAC ATCCTCAATG TGGCCTTCAC TATCATCTTC 240

ACCCTGGAGA TGATAAGCTC ATGGCCTTCA AGGAGGGCTA CTTTGGAGAC CCCTGGAATG" 300

TGTTTGACTT CCTGATTGTC ATTGGCAGCA TCATTGATGT CATCCTCAGT GAGATCGACG 360

ACCCAGATGA GAGTGCCCGC ATCTCCAGCG CCTTCTTCCG CCTGTTCCGT GTCATGAGGC 420

TGATCAAGCT GCTGAGCCGG GCAGAAGGAG TGCGAACCCT CCTGTGGACG TTCATCAAGT 480

CCTTCCAGGC CACCCTACGT GGCTCTGCTC ATCGTCATGC TCTTCTTCAT CTACGCTGTC 540

ATCGGCATGC AGATGTTTGG GAAGATCGCC TTGGTGGATG GGACCCAAAT AAACCGGAAC 600 AACAACTTCC AGACCTTCCC ACAAGCTGTG CTACTGCTCT TCAGGTGTGC AACAGGTGAG 660

GCCTGGCAGG AGATCCTACT GGCCTGCAGC TATGGGAAGC TGTGTGACCC AGAGTCGGAC 720

TATGCCCCAG GGGAGGAGTA CACATGTGGC ACCAACTTTG CATACTACTA CTTCATCAGC 780

TTCTACATGC TCTGTGCCTT CCTGGTCATC AACCTCTTTG TG 822

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 831 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(F) TISSUE TYPE: Small Cell Lung Carcinoma (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CGCAAACTCC ATAACCAATG CGTACAGTAT GCCCTGAAGG CCCGCCCACT GAGGTGTTAC 60

ATCCCCAAAA ACCCATACCA GTATCAGGTG TGGTATGTCG TCACTTCCTC CTACTTTGAA 120

TACCTGATGT TTGCTCTCAT CATGCTCAAT ACTATCTGCC TGGGCATGCA GCATTACAAC 180

CAGTCGGAAC AGATGAACCA CATCTCAGAC ATTCTCAACG TGGCTTTCAC CATCATCTTC 240

ACCCTGGAGA TGGTCTCAAA AGCTCATAGC TTTCAAGCCC AGGGGTATTT TGGAGATCCT 3σθ

TGGAATGTGT TTGACTTCCT AATCGTCATC GGCAGCATCA TTGACGTCAT CCTGAGCGAG 360 ATCGATGACC CAGATGAGAG CGCCCGTATC TCCAGTGCCT TCTTCCGCCT GTTCCGGGTC 420

ATGAGACTGG TCAAGCTGCT GAACCGGGCG GAGGGTGTGC GCACCCTGCT GTGGACATTC 480

ATCAAGTCCT TCCAGGCCCT GCCGTACGTG GCTTTGCTGA TCGTGATGCT CTTCTTCATC 540

TACGCGGTCA TCGGCATGCA GATGTTCGGG AAGATCGCCA TGGTGGACGG GACGCAGATA 600

AACCGGAACA ACAACTTCCA GACTTTCCCA CAGGCCGTGC TGCTGCTCTT CAGGTGTGCC 660

ACAGGGGAAG CCTGGCAAGA GATCCTGCTG GCCTGCAGCT ATGGGAAGCT GTGTGACCCG 720

GAGTCTGACT ACGCACCCGG GGAGGAGCAT ACATGCGGCA CCAACTTCGC CTACTACTAC 780

TTCATCAGTT TCTACATGCT CTGCGCCTTC CTGATCATCA ACCTCTTTGT G 831

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 249 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapien

(F) TISSUE TYPE: Small Cell Lung Carcinoma (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Trp Pro Cys Asx Val Gly Glx Pro lie Cys Ala Xaa Xaa Xaa Asp Xaa 1 5 10 15

Xaa Xaa Glu Pro Ser Asn Phe Xaa Glu Pro Phe Xaa Pro Arg Ser Xaa 20 25 30 Asn Xaa Xaa His Pro Xaa His Ala Xaa His Pro Xaa His Ala Trp Cys 35 40 45

Arg He Glu Arg Pro He Thr Cys His Pro Cys His Pro Leu Ala Ser 50 55 60

Glu Arg Glu Thr Ser Glu Arg He Glu Ser He He His Pro Leu Ala 65 70 75 80

Ser Glu He He Pro Arg Ser Xaa His Trp Xaa Xaa His Pro Xaa His 85 90 95

Ala His Pro His Pro His His His His His Pro Xaa His Pro Xaa His 100 105 110

His His Pro Xaa His Pro Xaa His Pro Xaa His Pro Xaa His His His 115 120 125

Pro Xaa Leu His Pro Xaa Ala Tyr Val Cys Leu Leu He Ala Met Leu 130 135 140

Phe Phe He Tyr Ala He He Gly Met Gin Val Phe Gly Asn He Gly 145 150 155 160

He Asp Val Glu Asp Glu Asp Ser Asp Glu Asp Glu Phe Gin He Thr 165 170 175

Glu His Asn Asn Phe Arg Thr Phe Phe Gin Ala Leu Met Leu Leu Phe 180 185 190

Arg Ser Ala Thr Gly Glu Ala Trp His Asn He Met Leu Ser Cys Leu 195 200 205 Ser Gly Lys Pro Cys Asp Lys Asn Ser Gly He Leu Thr Arg Glu Cys 210 215 220

Gly Asn Glu Phe Ala Tyr Phe Tyr Phe Val Ser Phe He Phe Leu Cys 225 230 235 240

Ser Phe Leu Met Leu Asn Leu Phe Val 245

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 406 amino acids

(B) TYPE: amino acid (D)^' TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: YES

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (F) TISSUE TYPE: Small Cell Lung Carcinoma ^• (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Trp Pro Cys Asx Val Gly Glx Pro He Cys Ala Xaa Xaa Xaa Asp Xaa 1 5 10 15

Xaa Xaa Glu Pro Ser Asn Phe Xaa Glu Pro Phe Xaa Pro Arg Ser Xaa 20 25 30

Asn Xaa Xaa His Pro Xaa His Ala Xaa His Pro Xaa His Ala Trp Cys 35 40 45

Arg He Glu Arg Pro He Thr Cys His Pro Cys His Pro Leu Ala Ser 50 55 60 Glu Arg Glu Thr Ser Glu Arg He Glu Ser He He His Pro Leu Ala 65 70 75 80

Ser Glu He He Pro Arg Ser Xaa His Trp Xaa Xaa His Pro Xaa His 85 90 95

Ala His Pro His Pro His His His His His Pro Xaa His Pro Xaa His 100 105 110

His His Pro Xaa His Pro Xaa His Pro Xaa His Pro Xaa His His His 115 120 125

Pro Xaa Leu His Pro Xaa Leu Gin Cys Val Gin Tyr Ala Leu Lys Ala 130 135 140

Arg Pro Leu Arg Cys Tyr He Pro Lys Asn Pro Tyr Gin Tyr Gin Val 145 150 155 160

Trp Tyr He Val Thr Ser Ser Tyr Phe Glu Tyr Leu Met Phe Ala Leu 165 170 175

He Met Leu Asn Thr He Cys Leu Gly Met Gin His Tyr Asn Gin Ser 180 185 190

Glu Gin Met Asn His He Ser Asp He Leu Asn Val Ala Phe Thr He 195 200 205

He Phe Thr Leu Glu Met Xaa Xaa Lys Leu Met Ala Phe Lys Glu Gly 210 215 220

Tyr Phe Gly Asp Pro Trp Asn Val Phe Asp Phe Leu He Val He Gly 225 230 235 240 Ser He He Asp Val He Leu Ser Glu He Asp Asp Pro Asp Glu Ser 245 250 255

Ala Arg He Ser Ser Ala Phe Phe Arg Leu Phe Arg Val Met Arg Leu 260 265 270

He Lys Leu Leu Ser Arg Ala Glu Gly Val Arg Thr Leu Leu Trp Thr 275 280 285

Phe He Lys Ser Phe Gin Ala Xaa Pro Tyr Val Ala Leu Leu He Val 290 295 300

Met Leu Phe Phe He Tyr Ala Val He Gly Met Gin Met Phe Gly Lys 305 310 315 320

He Ala Leu Val Asp Gly Thr Gin He Asn Arg Asn Asn Asn Phe Gin 325 330 335

Thr Phe Pro Gin Ala Val Leu Leu Leu Phe Arg Cys Ala Thr Gly Glu 340 345 350

Ala Trp Gin Glu He Leu Leu Ala Cys Ser Tyr Gly Lys Leu Cys Asp 355 360 365

Pro Glu Ser Asp Tyr Ala Pro Gly Glu Glu Tyr Thr Cys Gly Thr Asn 370 375 380

Phe Ala Tyr Tyr Tyr Phe He Ser Phe Tyr Met Leu Cys Ala Phe Leu 385 390 395 400

Val He Asn Leu Phe Val 405 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 407 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: YES

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(F) TISSUE TYPE: Small Cell Lung Carcinoma

(x) PUBLICATION INFORMATION: (A) AUTHORS: Tanabe,

(C) JOURNAL: Nature

(D) VOLUME: 328

(F) PAGES: 313-318

(G) DATE: 1987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Trp Pro Cys Asx Val Gly Glx Pro He Cys Ala Xaa Xaa Xaa Asp Xaa 1 5 10 15

Xaa Xaa Glu Pro Ser Asn Phe Xaa Glu Pro Phe Xaa Pro Arg Ser Xaa 20 25 30

Asn Xaa Xaa His Pro Xaa His Ala Xaa His Pro Xaa His Ala Trp Cys 35 40 45

Arg He Glu Arg Pro He Thr Cys His Pro Cys His Pro Leu Ala Ser 50 55 60

Glu Arg Glu Thr Ser Glu Arg He Glu Ser He He His Pro Leu Ala 65 70 75 80 Ser Glu He He Pro Arg Ser Xaa His Trp Xaa Xaa His Pro Xaa His 85 90 95

Ala His Pro His Pro His His His His His Pro Xaa His Pro Xaa His 100 105 110

His His Pro Xaa His Pro Xaa His Pro Xaa His Pro Xaa His His His 115 120 125

Pro Xaa Leu His Pro Xaa Leu Gin Cys Val Gin Tyr Ala Leu Lys Ala 130 135 140

Arg Pro Leu Arg Cys Tyr He Pro Lys Asn Pro Tyr Gin Tyr Gin Val 145 150 155 160

Trp Tyr Val Val Thr Ser Ser Tyr Phe Glu Tyr Leu Met Phe Ala Leu 165 170 175

He Met Leu Asn Thr He Cys Leu Gly Met Gin His Tyr Asn Gin Ser 180 185 190

Glu Gin Met Asn His He Ser Asp He Leu Asn Val Ala Phe Thr Tie 195 200 205

He Phe Thr Leu Glu Met Val Ser Lys Ala His Ser Phe Gin Ala Gin 210 215 220

Gly Tyr Phe Gly Asp Pro Trp Asn Val Phe Asp Phe Leu He Val He 225 230 235 240

Gly Ser He He Asp Val He Leu Ser Glu He Asp Asp Pro Asp Glu 245 250 255 Ser Ala Arg He Ser Ser Ala Phe Phe Arg Leu Phe Arg Val Met Arg 260 265 270

Leu Val Lys Leu Leu Asn Arg Ala Glu Gly Val Arg Thr Leu Leu Trp 275 280 285

Thr Phe He Lys Ser Phe Gin Ala Leu Pro Tyr Val Ala Leu Leu He 290 295 300

Val Met Leu Phe Phe He Tyr Ala Val He Gly Met Gin Met Phe Gly 305 310 315 320

Lys He Ala Met Val Asp Gly Thr Gin He Asn Arg Asn Asn Asn Phe 325 330 335

Gin Thr Phe Pro Gin Ala Val Leu Leu Leu Phe Arg Cys Ala Thr Gly 340 345 350

Glu Ala Trp Gin Glu He Leu Leu Ala Cys Ser Tyr Gly Lys Leu Cys 355 360 365

Asp Pro Glu Ser Asp Tyr Ala Pro Gly Glu Glu His Thr Cys Gly Thr 370 375 380

Asn Phe Ala Tyr Tyr Tyr Phe He Ser Phe Tyr Met Leu Cys Ala Phe 385 390 395 400

Leu He He Asn Leu Phe Val 405

Claims

1. An essentially pure, recombinant alpha-1 polypeptide subunit of the human voltage- sensitive calcium channel protein on human small cell lung carcinoma, or a portion thereof.

2. The polypeptide of claim 1, or a portion thereof, which is immunoreactive.

3. The polypeptide of claim 1 comprising the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:l.

4. The polypeptide of claim 1 comprising the amino acid sequence set forth in the Sequence Listing as SEQ ID N0:2.

5. The polypeptide of claim 1 comprising the amino acid sequence set forth in the Sequence Listing as SEQ ID N0:3.

6. A fusion protein comprising the polypeptide of claim 1.

7. An isolated nucleic acid having a nucleotide sequence encoding the polypeptide of claim 1.

8. The nucleic acid of claim 7, which is DNA.

9. The DNA of claim 8 having as at least a portion of its sequence the DNA sequence listed as SEQ ID NO:4.

10. The DNA of claim 8 having as at least a portion of its sequence the DNA sequence listed as SEQ ID NO:5.

11. The DNA of claim 8 having as at least a portion of its sequence the DNA sequence listed as SEQ ID

NO: 6.

12. An expression vector containing DNA encoding the fusion protein of claim 6.

13. An expression vector containing the DNA of claim 7.

14. A method of detecting and diagnosing small cell lung carcinoma in a human subject, comprising the steps of:

(a) providing an immunoabsorbent including a solid phase to which is attached a recombinant, immunoreactive alpha-1 polypeptide subunit of the human voltage-sensitive calcium channel protein on human small cell lung carcinoma, or an immunoreactive portion of the polypeptide;

(b) incubating the immunoabsorbent with a sample of a biological fluid, sampled from the subject, under conditions conducive for the binding of an anti-human calcium channel antibody to the immunoabsorbent-bound polypeptide;

(c) separating the immunoabsorbent from the sample; and (d) determining if the antibody has bound to the immunoabsorbent, the presence of the bound antibody being indicative of the presence of small cell lung carcinoma in the subject.

15. The method of claim 14 wherein the providing step (a) comprises providing an immunoabsorbent including a solid phase to which is attached a polypeptide having, as at least a portion of its amino acid sequence, the amino acid sequence listed as SEQ ID NO:l.

16. The method of claim 14 wherein the providing step (a) comprises providing an immunoabsorbent including a solid phase to which is attached a polypeptide having, as at least a portion of its amino acid sequence, the amino acid sequence listed as SEQ ID NO:2.

17. The method of claim 14 wherein the providing step (a) comprises providing an immunoabsorbent including a solid phase to which is attached a polypeptide having, as at least a portion of its amino acid sequence, the amino acid sequence listed as SEQ ID NO:3.

18. The method of claim 14 wherein the incubating step (b) comprises incubating the immunoabsorbent with a biological fluid selected from the group consisting of blood, serum, and plasma.

19. The method of claim 14 wherein the determining step (d) comprises:

(i) incubating the sample-treated immunoabsorbent with a labelled anti-human antibody, or a labelled human antibody binding portion thereof, under conditions conducive for the binding of the labelled anti-human antibody to an immunoabsorbent-bound human antibody; (ϋ) separating the immunoabsorbent from from unbound labelled anti-human antibody, or portion thereof, after the incubation period; and (iii) detecting the label associated with the immunoabsorbent as an indication of the presence of the anti-calcium channel antibody in the biological fluid.

20. A kit containing:

(a) an immunoabsorbent comprising a solid phase to which is attached an immunoreactive, recombinant alpha-1 polypeptide subunit of the human voltage-sensitive calcium channel protein on human small cell lung carcinoma, or an immunoreactive portion thereof; and (b) labelled anti-human antibody, or a human antibody-binding portion thereof.

21. The kit of claim 20 wherein the solid phase is attached to a polypeptide having, as at least a portion of its amino acid sequence, the sequence listed as SEQ ID NO:l.

22. The kit of claim 20 wherein the solid phase is attached to a polypeptide having, as at least a portion of its amino acid sequence, the sequence listed as SEQ ID NO:2.

23. The kit of claim 20 wherein the solid phase is attached to a polypeptide having, as at least a portion of its amino acid sequence, the sequence listed as SEQ ID NO:3.

24. The kit of claim 20 wherein the solid phase is selected from the group consisting of a bead, a microtitre plate well, and a test tube.

25. A method of treating Lambert-Eaton Syndrome in an afflicted human subject comprising the steps of:

(a) providing an immunoreactive, recombinant .alpha-1 polypeptide subunit of the human voltage-sensitive calcium channel protein on human small cell lung carcinoma, or an immunoreactive portion of the polypeptide; and

(b) administering the polypeptide, or portion thereof, with a pharmaceutically acceptable carrier, to the subject in an amount sufficient to block the binding of anti-calcium channel protein antibody to calcium channel protein on neurotransmitter- producing cells in the subject.

26. The method of claim 25 wherein the providing step comprises providing a polypeptide having, as at least a portion of its amino acid sequence, the sequence listed as SEQ ID NO:l.

27. The method of claim 25 wherein the providing step comprises providing a polypeptide having, as at least a portion of its amino acid sequence, the sequence listed as SEQ ID NO:2.

28. The method of claim 25 wherein the providing step comprises providing a polypeptide having, as at least a portion of its amino acid sequence, the sequence listed as SEQ ID NO:3.