GENOMICS 10,835-839 (1991)

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Assignment of the Human Gene for the cyl Subunit of the Cardiac DHP-Sensitive Ca2+ Channel (CCHLI Al)

to Chromosome 12pl2-pter PATRICIA A. POWERS,* RONALD G. GREGG,*+* PETER A. LALLEY,~ MARTHA LIAO, 11 AND KIRK HOGAN*

*Department of Anesthesiology, tDepartment of Pediatrics, and $Waisman Center on Mental Retardation and Development, University of Wisconsin, Madison, Wisconsin 53705; Kenter for Molecular Biology, Wayne Stare University,

Detroit, Michigan 48202; and (1 Department of Pediatrics, Eleanor Roosevelt Institute for Cancer Research, University of Colorado Health Science Center, Denver, Colorado

Received October 9, 1990; revised January 25, 1991

A human clone corresponding to the gene encoding the (Ye subunit of the cardiac dihydropyridine-sensitive calcium channel (CCHLlAl) has been isolated and partially se- quenced. Oligonucleotides based on the human sequence were constructed and used in the polymerase chain reac- tion to amplify specifically this human gene in human-ro- dent somatic cell hybrids. Using somatic cell hybrids that contained defined regions of human chromosome 12, the human CQ subunit of the cardiac dihydropyridine-sensitive calcium channel has been assigned to the short arm of chro- mosome 12 in the interval 12p12-pter. o 1991 Academic

Press, Inc.

Activation of voltage-sensitive calcium channels by membrane depolarization triggers key cellular re- sponses such as contraction, secretion, excitation, and electrical signaling (Hille, 1984). The L-type currents produced by voltage-sensitive calcium chan- nels are blocked by 1,4-dihydropyridine (DHP) deriv- atives, and thus the channels responsible for these currents are referred to as DHP-sensitive. These DHP-sensitive Ca2+ channels are present in a wide range of cell types (reviewed by Hess, 1990; Bean, 1989)) but have been studied most extensively in skele- tal muscle from which the channel has been purified. The skeletal muscle DHP-sensitive Ca2+ channel is a complex of five subunits, a,, aZ, /I, y, and 6 (reviewed by Catterall et aZ., 1988). The DHP-sensitive Ca2+ channels from cardiac muscle and the brain have pharmacological and electrophysiological properties that differ from those of the skeletal channel (Rosen- berg et al., 1986; Cognard et al., 1986; Takahashi and Catterall, 1987), suggesting that they may be struc-

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. M61130.

turally distinct entities; however, channels from all three tissues have very similar structural and immu- nochemical properties (Schmid et aZ., 1986; Cooper et al., 1987; Takahashi and Catterall, 1987, Ahlijanian et aZ., 1990), implying a conservation in their organiza- tion and a high degree of homology between their pri- mary sequences.

The primary amino acid sequences of four of the subunits (aI, a2, /?, and y) of the skeletal DHP-sensi- tive Ca2+ channel have been deduced from the DNA sequence of the corresponding cDNAs (Tanabe et al., 1987; Ellis et al., 1988; Ruth et al., 1989; Jay et al., 1990). The cDNA of the cardiac a1 subunit has also been isolated and sequenced (Mikami et al, 1989). The cardiac and skeletal aI subunit cDNAs share the same general organization and transmembrane topol- ogy but differ in their putative cytoplasmic regions. The predicted amino acid sequences of the skeletal and cardiac aI subunits show 66% overall identity and contain features characteristic of many other chan- nels (Tanabe et al., 1987; Ellis et al., 1988). Transfec- tion of full-length cDNA clones of the skeletal a1 sub- unit (Perez-Reyes et al., 1989) or the cardiac aI sub- unit (Tanabe et al., 1990) into fibroblast cell lines results in the synthesis of proteins with the electro- physiological and pharmacological characteristics of the DHP-sensitive Ca2+ channel. Messenger RNA de- rived from the cardiac DHP-sensitive Ca2’ channel cDNA is sufficient to direct the formation of a func- tional DHP-sensitive Ca2’ channel in Xenopus oo- cytes (Mikami et al, 1989).

The mouse muscular dysgenesis mutation (mdg; Pai, 1965a,b) is the consequence of the apparent ab- sence of the a, subunit of the skeletal DHP-sensitive Ca2+ channel (Knudson et al., 1989). The mutant phe- notype is rescued in cultured muscle cells from mdg/ mdg mice by microinjection of an expression plasmid

835 03&%7543/91 $3.00 Copyright 0 1991 by Academic Press, Inc.

All rights of reproduction in any form reserved.

836 SHORT COMMUNICATION

A

r GCTGCTGTACAGGGC IlIlIlIlII I I

h ggatccctggagcagtggtgccgtccttccgcagaggggaccctgcttctccagttccctctgtgggacctgtctcctcctgcagGCTGCTGTACCGCTC

r CATAGACTCCAACGAGGAGGACATGW;CCCCCGTTTAC~~CCGAGTGGAGATG~CATCTTCTTCATCATCTA~TCATCCTCATTGCCTTCTTCATG III IIIIII II III III1 IlIIIlI I IIIII IIII IIIIIlII IIIIIlIiIiIiIIIIIIIIIIIIIl IIll IIIlIllIIIIl

h CATCGACTCCCACACGC~G~CCCCATCTACAAGG

r ATGAACATCTTTGTGGGCTTTGTCATCGTCGT~CCAGG llllIlIIIII IIIIIIII IIIIIIIIlIIIII IIIIIIIlIIIIIII IIIIIIlIlIlIII IIIIlIIlIIIIlIIIII

h ATGAACATCTTCGTGGGCTTCGTCATCGTCACCTTTCAGGA~AG~~AGCAGGAGTAC~G~CTGT~~TGGAC~G~CCAGgtagcttcctagg

h aaggagcggaggccaagcggggcccacggagggaatggcagcctgcggcccaccccgcagaggggctgcgacagggagaggccgtgcagatactgagatc

r card. LLYRSIDSHTEDKGPIYNYRVEISIFFIIYIIIIAFFMMNIFVGFVIV~QEQGEQEYKNCELDKNQ 1216 lllllillllIlll1ll IIIIIlllllllllllllllll1~lll1lllllllllllllllllllll

human LLYRSIDSHTEDKGPIYKYRVEISIFFIIYIIIIAFFMMNFQEQGEQEYKNCELDKNQ IIII III II II I III IIIIIIlI IIIlIIIIIIIIIlIIIIIIII llIIIIIIlll

r skel. LLYRAIDSNEEDMGPVYNNRVEMAIFFIIYIILIAFFMMNIFVGFVIVTFQEQGETEYKNCELDKNQ 108.5

3293

100

3393

200

3480

300

400

FIG. 1. Comparison of the human DNA sequence with the rabbit 01~ subunit of the cardiac and skeletal DHP-sensitive Ca’+ channels. (A) Nucleotide sequence comparisons: The upper line (r) represents the rabbit skeletal aI cDNA (numbered as in Ref. (7)) and the lower line represents the human DNA sequence obtained in this study. Vertical lines connect identical nucleotides. The locations of the two oligonucle- otides used in the polymerase chain reaction are underscored. (B) Predicted amino acid sequence comparisons: The rabbit cardiac (I~ subunit sequence (upper line) is compared to the human sequence (center line) and the rabbit skeletal (Y, subunit sequence (lower line). Vertical lines connect identical amino acids. The numbering system is as in Mikamiet al. (14).

that carries the cDNA encoding the skeletal 01~ sub- unit (Tanabe et al., 1988). This work provided the first documented example of a genetic disease in ver- tebrates that is caused by a defect in the structural gene for an ion channel.

Localization of the genes encoding subunits of hu- man DHP-sensitive Ca2’ channel may direct us to identify genetic diseases that are the consequence of abnormal expression of subunits of this Ca2+ channel. Toward this aim, we cloned a segment of human geno- mic DNA that encodes part of the cardiac (Ye subunit of the DHP-sensitive Ca2’ channel and localized this gene to the distal portion of the p arm of chromo- some 12.

A fragment from the rabbit skeletal CQ subunit cDNA (pSKMcaCHal-7, provided by S. Ellis) was used as a probe to screen a cosmid library (Strata- gene). DNA fragments from the cosmids that we ob- tained were randomly subcloned into the phagemid vector pBluescript KS (Stratagene). Plasmids with inserts that hybridized to the rabbit cDNA probe, and therefore presumably contain exons of the human a1 subunit gene, were selected for further analysis. The DNA sequences of the inserts of these plasmids were determined by the chain termination method (Sanger et al., 1977) using a Sequenase kit (U.S. Biochemical) and double-stranded plasmid DNA as the template. DNA sequence analysis was performed using the GCG software (Devereaux et al., 1984) on a VAX computer.

Figure 1A shows the alignment of the rabbit skele- tal (Ye subunit cDNA (Ellis et al., 1988) with the hu- man genomic sequence. A 202-bp region of 88% ho- mology identified in this analysis presumably corre- sponds to an exon in the human gene. Consensus acceptor and donor splice sites are present in the hu- man sequence at the positions where the human geno- mic sequence and the rabbit cDNA sequence diverge, further supporting our conclusion that we have iden- tified an exon in the human (Ye subunit gene.

Figure 1B shows a comparison of the predicted translation product of the putative exon of the human (Y* subunit gene (middle line) with the amino acid sequences of the same region encoded by the rabbit cardiac (upper line) and skeletal (lower line) (Ye sub- unit genes. In this region, the human sequence differs from the rabbit cardiac (Ye subunit sequence by a single amino acid, at a position where the rabbit car- diac and skeletal sequences are identical; this proba- bly represents a substitution unique to the human se- quence. The human sequence differs from the rabbit skeletal LYE subunit sequence by 11 amino acids. At each of these 11 positions, the human sequence is identical to the rabbit cardiac sequence. We conclude that the sequence presented here contains an exon of CCHLlAl, the gene encoding the cardiac LYE subunit.

Oligonucleotides located in the exon (5’ TCGTG- GGCTTCGTCATCGTCA 3’) and in the intervening sequence (5’ GATCTCAGTATCTGCACGGCC 3’) were used as primers in a polymerase chain reaction

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(PCR) amplification (Sakai et al., 1985). Our se- quence predicts that the amplified product will be 190 bp in length. Polymerase chain reactions contained 250-500 ng of genomic DNA, 200 PM of each deoxy- nucleotide triphosphate, 1 PLM of each of the primers, 50 mM KCl, 1.5 mM MgCl,, 10 mM Tris-HCl, pH 8.3, 0.001% gelatin, and 1.25 units of Tuq DNA polymer- ase (Perkin-Elmer/Cetus) in a total volume of 50 ~1. The DNA was denatured for 2 min at 94”C, followed by 26 cycles of amplification with each cycle consist- ing of 45 s at 94°C (denaturation), 60 s at 65°C (an- nealing), and 2 min at 72°C (elongation). After the final cycle, an additional extension for 10 min at 72°C was performed. A lo-r1 aliquot of each PCR product was electrophoresed on a 1.5% agarose gel in 0.045 M Tris-borate, pH 8.3, 0.001 M EDTA. The amplifica- tion product was visualized by ethidium bromide staining under uv light. A single amplification prod- uct of 190 bp was observed when human genomic DNA was used as a template. No product was ob- served when the CHO cell line DNA or mouse geno- mic DNA was used as the template.

The results obtained from PCR analysis of 25 Chi- nese hamster-human hybrid cell line DNAs (pur- chased from the Bios Corp.) are summarized in Table 1. The amplification product was observed in all of the reactions in which the hybrid cell line contained human chromosome 12, and not observed in any reac- tions in which the hybrid cell line did not contain human chromosome 12. All of the other human chro- mosomes were excluded by at least two discordant hy- brids. These results establish that the human gene encoding the LYE subunit of the cardiac DHP-sensitive Ca*+ channel is located on chromosome 12.

We next analyzed a panel of somatic cell hybrids containing defined regions of human chromosome 12 (Law et aZ., 1986). The E4E cell line contains all of chromosome 12. Fll-1 and Fll-13 contain the long and short arms, respectively. Cell line 37A9 contains a short arm breakpoint at 12~12.05. Both cell lines 6-16 and 16-43 contain a long arm breakpoint, and 16-43 also contains a deletion of the distal end of 12~. The breakpoints of these hybrid cell lines and the re- sult of the PCR analysis using this hybrid panel are summarized in Fig. 2. Amplification product was ob- served with the following template DNAs: E4E, con- taining the intact chromosome 12; and Fll-13 and 16-16, each containing the short arm of chromosome 12. Amplification product was not observed with the following template DNAs: Fll-1, lacking the short arm of chromosome 12; 37A9, lacking the distal half of the short arm from 12~12.05 to the terminus; and 16-43, which lacks a portion of the short arm and a portion of the long arm. The location of the short arm breakpoint in 16-43 has not been established (Law et al., 1986); however, since this cell line expresses the

TABLE 1

Segregation of the Cardiac (Ye Subunit Gene of the DHP-Sensitive Calcium Channel with Human Chro- mosomes in Rodent-Human Somatic Cell Hybrids

DHP sequence/chromosome retention

Chromosome +/+ -/- +/- -/+ % Discordance

1 0 19 4 2 24 2 0 20 4 1 20 3 1 18 3 3 24 4 0 19 4 2 24 5 4 3 0 18 72 6 2 19 2 2 16 7 1 19 3 1 16 8 0 16 4 5 36 9 0 18 4 3 28

10 0 18 4 3 28 11 1 19 3 2 20 12 4 21 0 0 0 13 1 16 3 5 32 14 3 17 1 4 20 15 0 17 4 4 32 16 0 19 4 2 24 17 0 19 4 2 24 18 0 17 4 4 32 19 2 16 2 5 28 20 2 20 2 1 12 21 3 17 1 4 20 22 2 19 2 2 16 X 0 16 4 5 36 Y 3 20 1 1 a

Note. +/+ indicates that both the PCR product and the chromosome were present, -/- indicates that both the PCR product and the chromosome were absent, +/- indicates that the PCR product was present and the chromosome was absent, and -/+ indicates that the PCR product was absent and the chromosome was present. Numbers beneath each symbol indicate the number of cell lines that fall into each category. The percentage discordance is calculated as the sum of (+/-) and (-/+) divided by total number of cell lines examined.

human lactate dehydrogenase B (LDHB) isozyme (O’Connell et uZ., 1987), the breakpoint must be distal to the LDHB gene located at 12~12.1-12~12.2 (Roper and Craig, 1989). Using this hybrid panel, we assign the human gene encoding the (Ye subunit of the cardiac DHP-sensitive Ca*+ channel to the short arm of chro- mosome 12, distal to 12~12.1-12~12.2. More precise localization of this gene awaits the identification of additional somatic cell hybrids, in situ hybridization, or linkage analysis with polymorphic markers within or near this gene.

The complete absence of the (Ye subunit of the skele- tal DHP-sensitive Ca*+ channel causes muscular dys- genesis, a lethal condition in mouse (Pai, 1965a,b; Knudson et al., 1989). While it might be expected that the complete absence of the (Ye subunit of the cardiac DHP-sensitive Ca*+ channel would also produce a lethal condition, more subtle alterations in the struc-

838 SHORT COMMUNICATION

13.2 13.1 12.3

13.1

13.2 13.3

15

21.1 21.2

21.3

23

24.1 I I

24.2

24.31 24.32 24.33

FIG. 2. Breakpoints and PCR analysis of hybrid cell lines containing portions of human chromosome 12. Vertical bars represent the portion of human chromosome 12 present in each hybrid cell line. The PCR results are shown directly below the bar: lane 1, E4E contains an intact chromosome 12; lane 2, Fll-1 contains 12q; lane 3, Fll-13 contains 12~; lane 4,37A9 carries a deletion of p12.05-pter; lane 516-16 carries a deletion of q14.3-qter; lane 6,16-43 carries the same q14.3-qter deletion plus a second deletion in 12p (see text for discussion); lane 7 contains mouse genomic DNA; lane 8 contains human genomic DNA; and lane 9 contains HindIII-digested X DNA (Promega) and MspI-digested pBR322 DNA (New England Biolabs) as molecular weight standards.

ture or expression of the cq subunit of the cardiac DHP-sensitive Ca2+ channel may produce detectable cardiomyopathies. A search in McKusick (1990) for cardiomyopathic conditions reveals 65 entries. Some of these are known to be linked to chromosomes other than chromosome 12; however, many of these have not yet been mapped to any chromosome, and the involvement of the cardiac q subunit with any of these cardiomyopathies has yet to be established. The

cloning of the cardiac LYE subunit of the DHP-sensitive Ca2+ channel may provide a molecular tool that will facilitate the study of some of these disorders.

ACKNOWLEDGMENTS

This work was supported by a grant from the Department of Veterans Affairs to K.H. and by a grant from the Waisman Center on Mental Retardation and Development to R.G.G. K.H. is a recipi- ent of a B. B. Sarkey Anesthesia Advancement Award and a Foun-

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dation for Anesthesia Education and Research Young Investigator Award.

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