Molecular cloning of multiple subtypes of a novel rat brain isoform of the α1 subunit of the voltage-dependent calcium channel

Neuron,

Vol. 7, 35-44, July, 1991, Copyright

0 1991 by Cell Press

Molecular Cloning of Multiple Subtypes of a Novel Rat Brain lsoform of the al Subunit of the Voltage-Dependent Calcium Channel Anna Hui, Patrick T. Ellinor, Olga Krizanova, Jing-Jing Wang, Ronald 1. Diebold, and Arnold Schwartz Department University Cincinnati,

of Pharmacology and Cell Biophysics of Cincinnati College of Medicine Ohio 45267-0575

Summary Several cDNAs encoding an isoform of the al subunit of the voltage-dependent calcium channel were isolated from rat brain cDNA libraries. The complete nucleotide sequence of 6975 bp encodes a protein of 1634 amino acids, which corresponds to an M, of 186,968. The protein exhibits 71% and 76% homology to skeletal and cardiac al subunits, respectively. When compared with skeletal and cardiac al isoforms, the rat brain protein is intermediate in size at the amino terminus and shorter at the carboxyl terminus. Multiple subtypes of this al isoform cDNA were characterized. These are indicative of alternative splicing of a primary transcript and encode three variants between motif I and motif II and two within the S3 region of motif IV. Thus, multiple isoforms of this rat brain al subunit are possible.

of other cloned cation channels and contains the binding sites for the diverse class of drugs collectively known as the calcium antagonists (Naito et al., 1989; Striessnig et al., 1990). Additionally, the al subunit alone can function as a voltage-dependent calcium channel (Mikami et al., 1989; Perez-Reyes et al., 1989). The exact function of theother subunits is not known, although the skeletal a2 subunit has been shown to increase the current induced by the expression in oocytes of the cardiac al subunit (Mikami et al., 1989). Previously, we reported the partial sequence of a clone encoding a novel al isoform from brain (Koch et al., 1989). Subsequently, it has been shown that a heterogeneous family of calcium channels exists in brain (Snutch et al., 1990). Here we report the complete nucleotide and deduced amino acid structure of an isoform of the al subunit of thevoltage-,dependent calcium channel isolated from rat brain. We havealso characterized variants of this isoform that encode novel sequences within the cytoplasmic loop between motifs I and II and within the S3 domain of motif IV. These subtypes are indicative of alternative splicing of a primary transcript, a mechanism for the potential generation of multiple subtypes of this isoform of the a, subunit.

Introduction Results and Discussion Multiple types of voltage-dependent calcium channels coexist in excitable cells (Nowycky et al., 1985; Fedulova et al., 1985; Bean, 1989; Llinas et al., 1989). These calcium channels areessential for many cellular functions, such as musclecontraction, propagation of action potentials, maintenance of electrical activity, and neurotransmitter regulation. In neuronal tissue, four types of the voltage-dependent calcium channel -designated N, L, T, and P-have been classified according to their electrophysiological and pharmacological properties (Catterall, 1988; Bean, 1989; Hess, 1990). The L-type voltage-dependent calcium channel protein complexwas originally purified from rabbit skeletal muscle transverse tubules. This channel was thought to consist of five subunits: al, up, fi, 6, and y (Vaghy et al., 1987; Hofmann et al., 1987; Campbell et al., 1988; Glossmann and Striessnig, 1988; Vaghy et al., 1988), of which al, u2, p, and y have been cloned (McKenna et al., 1990; Tanabe et al., 1987; Ellis et al., 1988; Ruth et al., 1989; Jay et al., 1990). The so-called 6 subunit, however, is encoded by the a2 gene (De Jongh et al., 1990) and should now be regarded as a peptide linked to the a2 subunit (Jay et al., 1991). Recently the primary structures of the a1 subunits from rabbit cardiac muscle (Mikami et al., 1989; Slish et al., 1989), rat vascular smooth muscle (Koch et al., 1990), and rabbit lung (Biel et al., 1990) have been reported. In each case, the al subunit encodes a large hydrophobic polypeptide that has the characteristics

Isolation of cDNA Clones Encoding the Rat Brain lsoform of the al Subunit of the Voltage-Dependent Calcium Channel cDNA clones RB9 and RB19 were obtained by screening a rat brain cDNA plasmid library with the rabbit skeletal muscle clone hSKMCaCHal.3 (Ellis et al., 1988) as previously described (Koch et al., 1989). A Bglll-EcoRI fragment of RB9 was used to isolate clones RBII and RB48 from a rat hippocampal Igtll cDNA library. To isolate the 5’ end of the full-length clone, cDNA hSKMCaCHal.8 (Ellis et al., 1988) was used as a probe to isolate RB6. Because of the unusual nature of the 5’ end of this clone, a Sacll-Ncol fragment of RB6 was subsequently utilized to isolate clones RB517 and RB518. The alignment of these cDNAs is illustrated in Figure 1. Finally, a Sphl-PVul fragment of RB19 was used as a probe to isolate RB317. Three clones (1RB9, hRB19, and hRB317) were found that were identical at the 5’ end but then diverged to each encode a unique stop codon and 3’ untranslated region. To determine whether any of these clones represented the mature, fully spliced form of this transcript, we designed an oligonucleotide primer to the conserved 5’ region of the clones and a second set of primers that was in the unique, or putative 3’ untranslated region of each clone and used the primers in the polymerase chain reaction (PCR). After repeated trials with PCR amplification from rat brain mRNA, we were able to observe consistently the band corresponding

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to the hRB9 clone (Figure 28; Figure 7), but not to either of the other clones. This band was subcloned, sequenced, and found to encode a structure identical to hRB9. The clone, hRB9, is therefore the fully mature form of this transcript; clones hRB19 and hRB317 may be unspliced RNA messages, similar to those observed, for example, for other cloned cation channels (Loughney et al., 1989). Four overlapping cDNAs (RB6, RBII, RB48, and RB9) were used to constitute the initial full-length rat brain isoform. Clone RB6 contains a 526 bp 5’ untranslated region composed of an extremely GC-rich stretch followed by a predominantly AT-rich segment. The translation initiation site was assigned to the second in-

A

B

frame A7C; trlplet, since the nucleotide sequence surrounding this codon bears the closest resemblence to the eukaryotic consensus initiation site (Kozak, 1984). This reading frame was utilized since it encodes a sequence with significant similarity to other cloned calcium channels (Tanabe et al., 1987: Mikami et al., 1989; Naito et al., 1989). This frame remains open until a TAG termination codon is encountered at nucleotide position 5465 of RBa,. The complete RBa, clone consists of 1634 amino acids, with a calculated molecular mass of 186,968 daltons (Figure 3). Hydropathy plots (Kyte and Doolittle, 1982) and secondarystruc-ture prediction studies(Chou and Fasman, 1978) suggest that RBal has a topology similar to that of other cloned cation channels in that it consists of four internal, repeated units of homology (Figure 4). Each motif has five a-helical transmembrane segments (Sl, S2, S3, S5, and 56) and an amphipathic helix (S4) that contains positively charged residues at everv third or fourth position. This highly conserved S4 segment is presumed to act as the voltage sensor for voltage-dependent cation channels. Comparison of RBa, with the a, Subunits of the Voltage-Dependent Calcium Channel from Cardiac and Skeletal Muscle Figure 4 shows a comparison ot the amino acid sequencesot RBa, and the skeletal and heart a, isoform< previously reported. The overall amino acid identitv between RBa, and the rabbit skeletal and rabbit cardiac a, isotorms is 71%, and 76%, respectively. Differences found throughout the entire sequences of the brain, skeletal, and cardiac a, subunits indicate that each arise from a separate gene. This rat brain protein

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is similar to isoforms of other cloned cation channels in that the putative transmembrane regions of these isoforms are generally highly conserved, while divergence is localized to the amino and carboxyl termini as well as to the segments between the membranespanning regions. Sequence differences between the rat brain al protein and other al isoforms are found intheextracellularloops(between IS3and IS4; IS5and IS6; Ills5 and lllS6; IVS3 and IVS4), in the cytoplasmic loops (between IS6 and IISI; llS6 and IllSI), and in the amino terminus. The amino terminus of RBa, is intermediate in length when compared with the heart or skeletal sequences. The second half of this region (amino acids 50-125 of RBa,) bears homology to the heart and the aortic sequences, whereas the first 50 amino acids encoded by RBal are considerably different from either the skeletal or the heart protein. Indeed, the first 9 amino acids of RBa, consist of 6 adjacent methionine residues followed by 2 lysines and another methionine residue. Such an unusual amino terminus has not been reported for any other sequence and is suggestive of possible translational control of the expression of this message (Dr. Marilyn Kozak, personal communication). The carboxyl terminus of RBa, is considerably shorter than that of either the skeletal or the cardiac protein and is highly conserved among the three forms until 15 amino acids before the end of translation of RBa,. Overall striking similarities are observed between RBa, and the cardiac and skeletal al subunits in the proposed transmembrane region of each motif. Specifically, the S4 segment, which has been proposed to act as the voltage sensor for all voltage-dependent cation channels, has been nearly completely conserved among these isoforms. Interestingly, the S5 and S6 segments exhibit a similar high degree of homology among the isoforms, which is not observed among the Sl, S2, or S3 segments. Potential Regulatory Domains Encoded within RBa, According to the proposed protein topology of the rat brain al isoform, three potential N-glycosylation sites (Hubbard and Ivatt, 1981) are predicted to be on the proposed extracellular loops (Asn-154, -224, and -328). Of the three, Asn-154 and Asn-328 are conserved in all the isoforms examined, whereas the potential N-glycosylation site at residue224 is unique to the rat brain isoform.Afewdifferencesin potential regulatorysites exist between the RBa, protein and the other cloned a, isoforms. Four potential CAMP-dependent phosphorylation sites (Krebs and Beavo, 1989) are present in the proposed cytoplasmic regions of this protein (Ser-464, -848, -1489, and -1584). Ser-1584 is conserved in all a, isoforms, but the potential phosphorylation sites at Ser-464, -848, and -1489 are found only in this rat brain a1 isoform. The phosphorylation site present at Ser-687 of skeletal muscle al is absent in the brain isoform, and it is in the cardiac and aortic isoforms. This specific Serine residue, which is phosphorylated by protein kinase A in vitro, may be a regulation site

forthe a, subunit in skeletal muscle in viva (John et al., 1988). All of the a1 isoforms, including RBtx,, contain a putative EF hand calcium-binding domain (Babitch, 1990), within a highly conserved regiorl after IVS6 (amino acids 1463-1491 of RBa,). It is interesting that a putative phosphorylation site that is unique to this rat brain clone is located within this region. Northern Analysis of the RBal Transcript Poly(A)’ RNA from several rat tissues was used in a standard Northern blot analysis. A Pvul-Sphl fragment of RB9, which codes for a sequence in the region that is significantly homologous to other cloned calcium channels, hybridized to an 8.6 kb transcript in brain, heart, aorta, uterus, and lung and to a 6.5 kb band in aorta and skeletal muscle (Figure 5). Subsequently, a PCR product (PCRI) that encodes a sequence unique to RBal was used as a probe. One transcript of 6.5 kb was detected using mRNA isolated from rat brain (Figure 5). Previous references to an 8.6 kb transcript for brain (Koch et al., 1989,199O; Snutch et al., 1990) are most likely due to cardiac: or smooth muscle transcripts, since probes homologous to these tissues were employed in these hybridization experiments. Evidence for Alternative Splicing of RBa, Alternative splicing is a common mechanism for the generation of multiple isoforms from a single gene (Breitburt et al., 1987; Andreadis et al., 1987). Recent evidencefrom thecloning and expression of the products of the Shaker locus of Drosophila have shown that these alternatively spliced regions are responsible for the tissue specificity and/or the unique kinetic properties of many A-type potassium channels (for review see Jan and Jan, 1989). Other evidence has also been presented for alternative splicing of the sodium channel in Drosophila (Loughney et al., 1989) and recently of the L-type calcium channel in rat aorta (Koch et al., 1990). In the following sections we describe the characterization of two variable regions of RBa, (summarized in Figure 6) that are suggestive of alternative splicing of a primary transcript. The Third Membrane-Spanning Region of Motif IV Recently we have shown that two types of the cardiac and aorta al isoforms exist; these differ by a single, alternatively spliced exon encoding the S3 of motif IV (Kochetal.,1990).Asimilartypeof splicingisapparent in this brain isoform (Figure 6B). Two cDNAs, RB48 and RB9, were found to encode a similar, yet distinct sequence for the S3 of motif IV. Clone RB48 is identical in this region to clone RBD-55, a partial sequence recently reported by Snutch et al. (1990). All of the changes that occur within the S3 region are conservative;therefore,theoveralI hydrophobicityofthisa-helical membrane-spanning region is not altered. Additionally, the four negatively charged amino acids (3 aspartic acids and 1 glutamic acid) that have been postulated to be involved in voltage sensing and pore formation (Montal, 1990) are conserved. The two forms

CCGGATGTGAGCTCCGGCTGCCCGCGG~CCCGAGCCAGCGGCGGCGCGGGCGGCGA~~~C~GC~~~~~~~~A~~~~~~A~~~~~~~~~~~~~~~~~~A~~~~~GGCAIGGGGG~~G~G~C~ AGCGGCCCCGGCGGCCGGGCGGGCACCACCGCGGTGTCCCTCCGCTAGAGGAGGGGACCAAGCCAATTCTCCTTTGCAGCAAAAAAAGAGAAAAAAGAAAAAAAAGAAAAATTTACATG~ ATATATTACTAAGATAATATATACATCTGATTT~A~TTT~TACAAAAAG~TTATT~~GCTCCATTTTTCAAAAAAAGAAGGAGCGTGGGTGGCAGGCGGTTTTTATAAATTA~TCTTA~~ TTTGATTATTTGTCTCTGTCCTTCCCCACCCCCTGCTGAAGCGAAAATAAGGGCAGGGACCGCGGGCTCCTACCTATCAGTAACC~CCCATACCCGIT~CTCCCCCICC~TAACGCTGAG 490 500 51 I1 520 530 540 +/ 0 au 550 560 ,Y3 CATAGTGCCCTGCACACAGTAGGCGCTCAATAAATGTTCGTGGA~GATGATGATGATGATGATGAAAAAAATGCAGCATCAACGG~AGCAGCAACAGGACCACGCGAACGAGGCAAAC~A MHMMMMKKHOHOROOO~ I: H A Y ,

730 740 /SC 760 !/II ldli dill 790 800 tt.u CCAGACCATGAGCACGTCTGCACCCCLACCTGTAGGATCTCTCTCCCAAAGAAAACGlCAGCAAlACGCCAAGAGCAAAAAACAGGGiAA~l~~~i~lAACAGCCGACCIGCCCGiSCCC' OTMSTSAPPPVGSISOYKR~~OYAKSKK~GN',NSR~AQA 9 i,,

970 980 Y9C 1020 1000 1010 1030 1040 CATCCCGTTCCCTGAAGATGATlCTAA17CAACAAATCATAA~llGGAAAAAGTAGAATA~GCCTlCCTGATTATTTlTACAGlGGA~ACA~ I P F P E 0 D 8 N 5 T N H N, rKVFYAF, Il~TVli~

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1090 1100 !llO 1120 1130 1140 1150 1160 GCTGCATCCTAATGCTTACGTTAGGAACGGATGGAACTTACTGGATIT~GTTATAGTAATAG~AGGAl~GT~lAGlGTAAliliGGAAC~A L H P N A Y V R N G W N, I[1 i V IV IV G, ISVII':

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610 620 630 640 650 660 670 680 691! TGCAAGAGGCACCAGACTCCCTAT~~C~GGTGAAGGACCAACT~CTCAGCCGAACAGCTC:AAGCAAGCAAACCGTCCTGTCTTGGCAAGC~G~~\,~~ ARGTRI PISGFGPlSOPNSSX~TVtSWOhP'

850 860 8/O 860 690 900 910 920 CTTCTGTTTATCCCTCAATAACCCCATCCGAAGAGCCTGCATTAG~ATAGTGAATTGGAAACCA~TTGACATATTTATATTA~TGGC~A~I FCL5LNNPIRRACIS:VNWKPrDlrl!lA!~ANCV4lA"

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4: 1 c.?:, 1330 1340 :35:1 :1!3 :383 1400 1360 ;393 AGCCATGGTCCCCCTCCTCCACATAGCCCTTGGTGGTGTTATlCGTAAlTAlCATCTATGCGAl~ATAGGGTIGGAACTTTTTATTGGAAAAAlGCACAAAA~C~Gl~iTlTTG:lGAi~: IG L i t F : G ," 1 YT AMVPLtHIALtVL'V!II " A I

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1450 1460 -413 :499 1500 1520 ,:)L .540 1480 li:3 .,>a AGATATAGTAGCTGAAGAGGACCCAGCTCCATGCGCGTTCTCAGGGAATGGACGCCAGTGT~CC~CCAA~GGCACGGAATGTAGGAGCGG~'~G~TCGGCCCCAA~GGAGGCAT~A~T~A DIVAEEDPA~CAFSGVGROCAANGTECRS~WVGP~GG:‘Y

Sk:

1570 1580 11:3 162:: ibiL .66ii .bii: !!J93 1600 163C 1640 TTTTGACAACTTTGCCTTTGCCATGCTCACTGTGTTCCAGTGCATCACCATGGAGGGClGGACAGATGlGCTCTACTGGGTTAA~GATGC~A'A~GATGGGAGlGG~CATGGG~GlAT~~ FDNFAFAM, 1 VFOC:-MEGW 'OVLVWVNDA.;WrW"WV'

h8,

:74c 1690 1700 1713 :ii0 1760 Iii; i/8C .!4U 1110 1720 TGTTAGTCTGATCATCCTTGGCTCATTTTTCGTCCTTAACC~GGTTC~~GG~GTCCT~AG~GGAGAA~TCTCAAAGGAAAGAGAGAAGGCIAAAGCACGTGGAGAT~TCCAGAAGC~~~~ VSLIILGSFFVLNLV' G V L S G FFSKFRFKA,ARG?:"<

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1810 1620 l8iU iii60 1880 iaYi 3ui * ,J 183C 1840 :870 GGAGAAGCAGCAGCTGGAAGAGGATCTCAAGGGCTACTTGGACTGGATCACCCAGGCAGAAGACATAGATCCTGAGAATGAAGAGGAGGGlG~A~AGGAGGGCAAA~~AAACAtiASCA. EKO~LEEOLKGYLOW:T~A~~lDP~~EF~~~~ IGYYY'

,i;

1930 1940 .3/c i980 19ic 1960 :990 2300 GCCCACCAGTGAGACCGAATCTGTGAACACAGAGAACGTAAGTGGTGAAGGCGAGACCCAGGGA?GCTGTGGAAGTCTCTGCCAAGC~A PTSETESVNTENVSGIGETOGC CGSLCOA.'YSK

iZ,L

2050 2060 2093 2lOC 20/o 2080 2110 2120 TCGCTGGAACCGGTTCAATCGCAGGAGATGCAGGGCAGCTGTGAAG~~TGTCACGT~lTA~~GGClGGlGATCGlTCTGGTGli'CT~AA~A, RWNRFNRRRCRAAVKSVTr " w 1 v I v I v

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2410 2420 2430 2440 2433 246i 2480 2470 CTTCAAAGTGACCAGGCACTGGACTTCCCTGAGCAACTTGGTGGCGTCC~TGlTAAACTCCAlGAAGTCCATCGCCTCTCTGCTGTT~~-::i.:' FKVTRHWTS SNLVAZ. LNCMKSIASL!

143,

2530 2540 7113 2580 2550 2560 2590 2600 GCTGGGGATGCAGCTGTTCGGAGGGAAGTiCAATTTCGATGAGACACAGACCAAGCGAAGCA~ClTCGACAACTlCCCACAG~CG~l~~ LGHOLFGGK'NFCIFTOT KRS-FDNZPZA'

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2650 2660 267C 2680 269C Z/UC 21lC CTGGAATGCGGTGATGTATGACGGCATCATGGCCTACGGAGGC~CGICCICCTCTGGAAlGA~TGlCTGTATTTACTTCAlCAit~~ WNAVHYDGIYAYGGPSSSGr:VCIYFll'

/

7720

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2690 2900 ZY!C 2920 7430 2Y4C 2Y5C 2960 AAAAAACAACAAACCAGAGGTCAACCAGAIAGCCAACAGTGACAACAAGG~~ACTAT~GA~GA~~ATCAAGAGGAGGCTGAAGA~AAGGA~L, K N N K P E V N 3,A N S D N K V T ,I: D" 0 E F A C D

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3050

306C

307c

3080

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3130 3140 3150 3160 31/c 3180 3190 3200 CTTCATTCTTAGCAAGACCAACCCGATCCGTGTGGGTTGCCACAAACTCA.CAACCACCACATCITCAC~AACCTCATCCTGGTCTT(A FILSKTNPIRVGCHK~INHYIFT~LILV~'M

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2770 2780 28lG L62C Z/SC 2800 2630 2840 CTTCTTGGCCATTGCTGTAGACAA~TTGGCTGATGCTGAAAGTCTGAACACTGCTCAGAAAGAGGAAGCCGAAGAAAAAGAAAGGAAAAA~,~ FLAIAVDU!ADAES,N'ACK[ F A E i K E R

3010 3020 3UiO 3040 TGAAGAGGAAGAGGAGGAGGAAGAGGACGAGCCTGAGGIT EEEEEEEEOEPEVPAC

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3250 3260 3210 3280 3290 33cu 3310 3320 32,x 334C GGATCCCATCCGCAGCCACTCCTTCCGCAACACTATACTGGGCTAC~TTGACTATGCTTTCACAGCCATCTTTACGGTTGAAA~~CT~~~AAA~A~GA~AAC~~~~GGAGCC~ DPIRSHSFRNTILGYFDYAFTA,FTV~!'~~UTT-~A:

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Multiple 39

lsoforms

3370

of a Rat Brain Calcium

3380

3390

3400

Channel

3410

3420

3430

3440

3450

3460

3470

3480

CAAAGGGGCTTTCTGTAGGAACTACTTCAATTTGCTGGACATGCTGGTCGTTGGGGTGTCTCTGGTGTCATTTGGGATTCAATCCAGTGCCATCTCGGTTGTGAAGATTCTGAGGGTCTT KGAFCRNYFNLLDMLVVGVSLVSFGIOSSAISVVKILRVL 3490 3500 3510 3520 3530 3540 3550 3560 3570 3580 3590 3600 AAGGGTCTTGAGGCCTCTCAGAGCAATCAACAGAGCAAAAGGACTTAAGCACGTGGTCCAGTGTGTCTTTGTGGCCATCCGAACCATCGGCAACATCATGATCGTCACGACCCTGCTCCA RVLRPLRAINRAKGLKHVVDCVFVAIR?IGNIMIVTTLLO 3610 3620 3630 3640 3650 3660 3670 3680 3690 3700 3710 3720 GTTCATGTTTGCTTGCATTGGGGTCCAGCTGTTCAAGGGGAAGTTCTACCGTTGCACAGATGAGGCCAAAAGTAACCCCGAGGAGTGCAGGGGGCTTTTCATCCTTTATAAGGACGGCGA FMFACIGVOLFKGKFYRCTDEAKSNPEECRGLFILYKDGD

3730

3740

3750

3760

3770

3780

3790

3800

3810

3820

3830

3840

TGTCGACAGTCCCGTGGTCCGTGAGAGGATCTGGCAAAACAGTGATTTCAATTTCGACAATGTCCTTTCGGCTATGATGGCGCTCTTCACGGTCTCGACTTTTGAGGGCTGGCCCGCGTT VDSPVVRERIWONSOFNFDNVtSAtlMALFTVSTFEGWPAt

3850

3860

3870

3880

3890

3900

3910

3920

3930

3940

3950

3960

GCTGTACAAAGCTATCGACTCAAACGGAGAGAACGTTGGTCCTGTCTACAACTACCGTGTGGAGATCTCCATCTTCTTCATTATCTACATCATCATCGTGGCCTTCTTCATGATGAATAT tYKAIDSNGENVGPVYNYRVEISIFFIIYIIIVAFFMMNI

3970

3980

3990

4000

4010

4020

4030

4040

4050

4060

4070

4080

CTTCGTGGGCTTCGTCATCGTCACCTTCCAGGAGCAGGGAGAAAAGGAGTATAAGAACTGTGAGCTGGACAAAAATCAGCGTCAGTGTGTGGAATATGCCTTGAAGGCCCGCCCCTTAAG FVGFVIVTFOEOGEKEYKNCELDKNQROCVEYALKARPLR

4160 4170 4180 4190 4200 4090 4100 4110 4120 4130 4140 4150 GAGATACATCCCCAAAAACCCATACCAGTACAAGTTCTGGTACGTGGTGAACTCCTCGCCTTTCGAATA~ATGATGTTTGTCCTCATCATGCTCAACACGCTCTGCCTGGCCATGCAGCA RVIPKNPYDYKFWYVVNSSPFEYMMFVLIMLNTLCLAMDH 4210

4220

4230

4240

4250

4260

4270

4280

4290

4300

4310

4320

CTATGAGCAATCCAAGATGTTCAATGACGCCATGGACATTCTGAACATGGTCTTCACGGGGGTCTTCACCGTTGAGATGGTTTTGAAAGTCATCGCATTTAAGCCCAAGGGGTATTTTAG YEOSKMFNDAMDILNHVFTGVFTVEMVLKVIAFKPKGYFS

4330

4340

4350

4360

4370

4380

4390

4400

4410

4420

4430

4440

TGACGCCTGGAACACGTTTGACTCCCTCATCGTAATCGGCAGCATTATAGACGTGGCACTCAGCGAAGCTGACCCATCTGACAGTGAGAATATCCCTCTCCCAACTGCCACACCTGGGAA DAUNTFDSLIVIGSIIDVALSEADPSDSENIP~PTATPGN

4450

4460

4470

4480

4490

4500

4510

4520

4530

4540

4550

4560

CTCTGAAGAGAGCAATAGAATCTCCATCACCTTTTTCCGTCTTTTCCGAGTGATGCGGTTGGTGAAGCTTCTCAGCAGAGGGGAAGGCATCCGGACTCTGCTATGGACCTTCATTAAGTC SEESNRISITFFRLFRVMRLVKttSRGEGIRTLLUTFIKS

4570

4580

4590

4600

4610

4620

4630

4640

4650

4660

4670

4680

CTTCCAGGCACTCCCATATGTCGCCCTCCTCATTGCCATGCTCTTCTTCATCTACGCCGTCATTGGCATGCAGATGTTTGGGAAGGTTGCCATGAGAGATAACAACCAGATCAATAGGAA FOALPYVALLIAMLFFIYAVIGMDMFGKVAMRDNNOINRN 4690 4700 4710 4720 4730 4740 4750 4760 4770 4780 4790 4800 CAACAACTTCCAGACGTTTCCCCAGGCAGTGCTGCTGCTCTTCAGGTGTGCAACAGGGGAGGCCTGGCAGGAGATCATGCTCGCCTGCCTCCCTGGGAAGCTGTGTGACCCGGACTCAGA NNFDTFPOAVLLLFRCATGEAUQEIMLACLPGKLCDPDSL!

4810

4820

4830

4840

4850

4860

4870

4880

4890

4900

4910

4920

TTACAACCCAGGAGAGGAATATACTTGTGGGAGCAACTTTGCCATTGTCTACTTCATCAGCTTTTACATGCTCTGTGCGTTCCTGATCATCAACCTCTTCGTGGCTGTCATCATGGACAA YNPGEEYTCGSNFAIVYFISFYMLCAFLI INLFVAVIMDN 4930 4940 4950 4960 4970 4980 4990 5000 5010 5020 5030 5040 TTTTGACTATCTGACGAGGGACTGGTCTATTCTGGGGCCTCACCACTTGGACGAATTCAAAAGAATATGGTCTGAATATGACCCCGAGGCAAAGGGAAGGATAAAACACCTTGATGTGGT FDYLTRDWSILGPHHLDEFKRIWSEYDPEAKGRIKHLDVV

5050 5060 5070 5080 5090 5100 5110 5120 5130 5140 5150 5160 CACTTTGCTCCGACGTATCCAGCCTCCCCTGGGATTTGGAAAGTTGTGTCCACACCGAGTGGCGTGTAAGAGATTGGTCGCCATGAACATGCCTCTCAACAGTGATGGGACAGTCATGTT TLLRRIOPPLGFGKLCPHRVACKRtVAMNtlPtNSDGTVMF 5170 5180 5190 5200 5210 5220 5230 5240 5250 5260 5270 5280 CAATGCAACTCTGTTTGCTTTGGTACGGACGGCTCTCAAGATCAAGACTGAAGGCAACCTGGAGCAAGCTAATGAAGAGCTCCGCGCTGTGATAAAGAAAATCTGGAAGAAGACAAGCAT NATLFALVRTALKIKTEGNLEoANEELRAViKKIWKKTSN 5290 5300 5310 5320 5330 5340 5350 5360 5370 5380 5390 5400 GAAGCTACTTGACCAAGTTGTCCCTCCAGCTGGTGATGATGAGGTAACCGTGGGGAAGTTCTATGCCACTTTCCTGATACAGGACTACTTTAGGAAATTCAAGAAGCGGAAAGAGCAAGG KLLDDVVPPAGDDEVTVGKFYATFLIODYFRKFKKRKEQG

5410

5420

5430

5440

5450

5460

5470

5480

5490

5500

5510

5520

CCTGGTGGGGAAATACCCTGCGAAGAACACCACGATCGCCCTACAGATGCTTGAACGGATGCTCTAGAGTTGTCTGCCTGAGCTACCGCACCAAGCTGGTTAGTCCGGAGGCGCTGTGGC LVGKYPAKNTTIALOMLERML' TAAGGCCTTGAAAGGGCAAACTCCCCCTCGAGCTTATTTCTACTGAAGCAGCATTTCAGGAGAAATGCAGGCACCAAAGCCCTGGGGAGACCCCTTGCGAGCAAGGCACTCCTCCGGAAG GAAGGAAGGAAGGCAGAAGCCGCCGCTGTGTGTGGCTCATCTGTGGAGCCCTGGCTGCCTGTTGCGGACACTGCTCATGGTGGCTCCTTTTCATGAAAGGGAACATATGAGTTATGTTTT CCCCTTCCAAGCCAGAGACTCAGTGAGAGGGAAAGAAAGAGAGAGAGGGAGGGAGAGAGGGAGAGAGAGAACGCAAAGGGGCAGCCACCCCACAGAGAAATCAAATGAACACAACTTGCT GACGATGGCCAGTGAATAAAATAAATAATGTTTTTAATATATATATATATACATATATATGTGTATGTATATATATATGACTAAGCAGACCTGTGTAAGGCAGACAACGGACTCTGCCCC AAAGTCCTGCATCCCTAGTTTGCCCGGACTCCCAAAGACTGGCAGGCTGATTGGAACTGAGCAGACAGAGTTCCTTTCCTGTGCCAGCACGTCACAACCTCCTGACCACCCGGCCCTGCT TGCTGCAGTGACCTAGTCCAGAATGCACTGTAGGTGGAAGGCCAGGAAGCTCAGACACACCCATTGAAACTAACACATACACCCGCATGCCAAAACCAACCAGGCAACAGCTCAGGTTCC GTCTCCCCTACCGTGTCCGCGGGGAAACGCCACCACACCCAAACCTCATCTACCATTGTTCGTCTCTAGCCCGCACTCAGGACCAGTCTGGGACGCTCTTTGGAAGCAGTGTGGTATAGT TTCAAGGACACTGGGAGTCAGGGAACCTGGGTTCTAGTCCCAGCTCAGCCATTCACTTGCTGCGTGACCTTGGGCAAGACACTTAACCTCTCTGTGCCTCAGTTTCCCCAACTGTAAAAG GGGGTAATAATGTCGACCTACCTCACAGGGCTGTTGTGAGGAATCGCTACGTGACTGTAAAGGCTCCTTGGACGTATAATTGCTTATTATTAAGACTTCAACATTAATAATATCATATGC CTGTTTACTACCAGAACTTCAAGAAAGTCTTGGTTTGATCTCTTTTCTGTTCTGTAATGTAGTTATCCATCGAGAATTCTCCCCCCTTTTAAGATCCTAGGCAATGCATAAAGATGTAAG CAGGTAATAGCCAGACCACGTTTCCAAGTAGTAACAATAGGAGATTTTTGACCAAAACGTTGGTCGATGAGGGCTTTGCTGTCAGATTCTTAACACCTCCTCAGAGGGGGTCGAGTCTTA ACCTCTGAAGTTAGTCTACGATGCCCAAAATGGCCAAGGTAGGAGAGTCAGTATTCAAAGTTTGGGGGACGAATCTAAAAAAAGAAGAGGCAAAGGAAAAGGAGAGGAAGAAGGTGGGGG GGCTTCAATTCTTGATCT

Figure 3. Nucleotide The nucleotides

Sequence

are numbered

and Deduced

Amino

Acid Sequence

at the top of the sequence

for Rat Brain (x1

and begin with

from the brain and aorta aI isoforms share a noticeable degree of similarity between each class (RB9 and VSMa, versus RB48 and VSMa,.,), yet between the brain subtypes (RB9 versus RB48) there is a higher degree of similarity than between the cardiac subtypes (VSMa, versus VSMa4.

the first nucleotide

of the cDNA.

It has recently been shown in our laboratory that the two subtypes of the cardiac al are generated by the mutually exclusive splicing of these two exons from a single transcript (Koch et al., 1990; and unpublished data). Given the similarity between the cardiac and brain subtypes, this would be evidence that this

Brain Heart skeletal

----------~~~ -~-----------WMH~~~KK~OHL)ROOOEDHANEANVARGTRLPISGEGPTS~PNSSK~TVLS~OAAIDAAROAKAA~T~STSAPPPVGS~SORKROOYAKS HLRALV~PATPAVOPtPSHLSAETESTCKGTVVHEA~LNHFVISPGGSNVGSPRPAHANHNANAAAGLAPEHIPTPGAA~~~~~~~~~~~~~~-LMGSAGN~TISTV~ST~~~~~~~G~P ..~~...........~ .~................~...__.........._....__.._............ ~~nfp.s.arx.

IS1 Bra,”

I..Y.-

IS2

-P

IS3

Heart Skelet.3,

KKQGNSSNSRPARAtFCLSLNNPlRRACISIVN~KPFDIFILLAlFANCVALAIVIPFPEDDSNSTNHNLEKVEVAFLIIFTVETFLKIlAVGLLLHPNAVVRNGUNLLDFVIViVGtFS . . . . . . . . . ..*..S...R....~........~...~......~.....~............~..... . . ..STT~T..P...L..T.K...........E....E.....T......... ..PLPEVLP..P......T.a..L.K......E....ET,...T.........V"L.~....N..L.LG...~..F..TV.S,.~~M......F.F.~~..~.S..."...,..FL.".~

Bra,”

-VILE~LTKETEGGNHSSGKSGGFOYKALRAFRVLRPLRLVSGVPSt~VVLNSIIKA~VPLLHIALLVLFVIIIVAIIGtCLFIGK~HKTCFFADS-~DIV-

IS5

Is4

EDPAPCAFS-GNG AL . . .."NaEGVA.VP.......D..S...LET.H. .TVEN.K.F...RT-.5.

tlea rt Sk,,leta,

*....*...*D.A.*LG..G~.,..........................................................."... ~...."NV,aSNT*P~.S.G~.L..............................F...L..F........~"...........K......."",GT.....

Brain Heart Skeletal

RQCAANGTECRSGYVGPNGGlTNFDNFAFA~LTVF~CITMEG~T~VLV~VNDAIG~E~P~VVFVSLIlLGSFFVLNLVLGVLSGEFSKEREKAKARGDF~KLREK~~tEEDLKGVtD~lT . . . . . . . . . . . . . . . . . . . . . . . ..na..n.y.L . . . . . . ..y.F................................................... . . ..a...v.Kp..D..KH..... . . . . . ..T.......S..T..............R..~s... .P.TI..S...G..P..."...H....G.S...."....................N....,...T..L.....,..,

Brain Heart Skeletal

OAEDIDPENEEEGGEEGK-RNTSWPTSETESVNTENVSGEGET~GCCGS-LC~AISKSKLS-~-RR~RRUNRFNRRRCRAAVKSVTFVULVIVLVFLNTLTlSSEHVN~PDULT~IODi~ . . . . . . . . ..D....D.E.p..H . . . . . . . . . . . . . ..*.GDIEGEN..*R.IHR..... F.....V.......C..K.......NV......F.........~......."...E"..T. .G."".----V.DLR...-LSLEEGG.D..---. ..~..~ ~~-..."EIEGLN.,,aF,.H..a...VF.~K."DL...RV......L,.*....S.*..."...,...HL....

BMl”

NKVLLALFTCE~LVKMVSLGL~AVFVSLFNRFDCFVVCGGITETILVELELHSPLGVSVFRCVRLLRIFKVTRHUTSLSNLVASLLNS~KSIASLLLLLFLFIIlFSLLG~~LFGGKFNf ..~......A...L.....................,.....L......TKV . . . . . . ..L..........I..".N............"R.............................. . . . . . . . ..R................*.........R"g. .R...S...,...L...G...Ra..H.,..........S..L.LL...SGA.T...,..L..,....L..,.K"....

Is6

US1

IISZ Heart Skeletal

Brain

us3

1IS5

us4

Heart Skeletal

DETOTKRSTFDNFP~ALLTVFOILTGED~NAVnYDGlHAYGGPSSSG~IVCIVFllLFICGNVILlKLFLAIAVDNLADAESLNTAOKEEAEEKERKKIAR ..~..R.........S..............S.............FP..L.................NV...............TS.....E ED.EVR..N........,S...V.......S...N.........vp.VL.........V......~NV......-.~.i....~S...~K...~~.R.~S.GLp~KT~~~.Sv~~K.

KEELENK~

Brain tkert Skeletal

~--------KNNKPEVN~IANSDN-----KVTlDDVQE-EAEDKDPVPPCDVPVGEEEEEEEEDEPEVPAGPRPRR~SEtN~KEKIAPlP~GSAFFILSKTNPIRVGCHKLINHHlFTNt . ..~.".....PL..."L...*V.~..*.....F.PN.RF.La..R.V.DT..... VGKPALEEA.EE.I.LKS.TADGESPPTT.INW..L.PN.S...S..-----.NP.TTG..D.E . . . . . . . ..LEa..KGEG.PTT~K.....LKVDEFESN.VN.V.....S~.F......GDD... . . . ..VS....PL*..~L...A"....*.S...F.P..KV..L..R~".~TY...F

Brain Heart Skeletal

ILVFIWLSSAALAAEDPIRSHSFRNTlLGVFDVAFTAIFTVEILLKUTTFGAFLHKGAFCRNVFNLLD~LVVGVSLVSFGI~SSAISVVKILRVLRVLRPtRAINRAKGtKHVV~CVFVA ..F..L...,S......Va"T....H..F...,V..T... ,..*....~y.......s......., ..L...S...,.........N...............~................. ..L..L.............*E.v..Q...... ,...sy.....y.....y.......s....... ,..~...*...,.~GLE..T.,..............

Braill Heart Skeletal

IRTIGNIHlVTTLLOF~FACIGVOLFKGKFVRCTDEAKSNPEECRGLFILVKDGDVDSPVVRERIY(INSDFNFDNVLSAH~ALFTVSTFEGUPALLVKAIDSNGENVGPVVNVRVEI~ . . . . . ..y..................... L.T.S.SS.aTEA..K.N".T....E..H.,,aP.S.E..K.D...V.A...............E...RS..."T.DK..,.......... . . . . . ..VL.....................FS.N.LS.~TE....."""". . . ..pT~~EL.p.~..~N..~...V.....~.,.........~...~.....~.~~....,~,.,~L..

Brain Heart Skeletal

FIIVIIIVAFFMHNIFVGFVIVTFOEOGEKEVKNCELDKN~R~CVEVAtKARPLRRVlPKNPV~VKFUVVVNSSPFEVMHFVLIHLNTLCLAM~HVE~SK~FNDAHDltNHVFTGVFTVE . . . . . . , . . ..'...'.'..........~...............................~"... V......T"...L.....L...,.......G..CL.K,..N....L.,.L.... . . . . ..L~.....................T...............a...... . ..C.........~V....~.."...~..~......,..G...."..EE~.~,S....VI,,,,..!.

. . . . . ..L..~........T*.p.K.~EV

-

IIISl

Ins2

IIIs.3

IIIS4 ,......

IIIS5

TVS3

--

IVs4

Ns5

Braill Heart Skeletal

HVLKVIAFKPKGYFSDAUNTFDSLIV~GSIIDVALSEADPSDSENIPLP~ATPGNSE--~-ESN-R~SITFFRLFRVMRLVKLLSRGEGIRTLLU~FIKSFOA~PVVALLIAMLFFIYAV .,..~...........P.."..F..........,...TN.....~,E"TaCSPS~.~......NS ............................................ .... .,..LL...~R...G.P.."..F..........,...,.TFL~SSGG."CLGG.CGN"D~D..~....SA ,.....*.." .............

Brain Heart Skeletal

IG~Q~FGKVA~RDNN~INRNNNF~TFPDAVLLLFRCATGEAU~EI~LACLPGKLCDPDSDVNPGEEVT-~CGSNFAIVVFISFV~LCAFLIINLFVAVI~DNFDVLTRDWSiLGPHHL?E . . ..V.....LN.TTE...........................D.....~...K.A.E.EP"NST.GETP... S..VF.......................................... . . . . . . . . ..LV.GT..............................L...S" . . . . ..E...A.......~..T..."". ..__._.........t_....._.......

Brain Heart Skeletal

FKRlWSEVDPEAKGRlKHLDVVTLLRRI~PPLGFGKLCPHRVACKRtVA~N~PLNSDGTV~FNATLFALVRTALKlKTEGNLE~ANEELRAVIKkI~KKTS~KttD~VVPPAGDDEVTVG . . . ..~..........................................S.........................~.......................~..................... ..*..*..............................F...........G...........T....,........... ..F.........,...,..R.........,..,........

Brain tlea rt Skeletal

KFVATFLIODVFRKFKKRKEOGLVGKVPAKNTTIALQMLERML* . . . . . . . ..E................-.SaRNALS..*GL.T.H . . . EH....M..a.E-VV.-.RP.KD.VaI.AGL.TIE

..v ........ ." ........

NS6

Figure 4. Amino

Acid Similarity

(477

residues)

GVSSL*

(306

residues)

IPPRP+

._....,

Comparison

The primary amino acid sequence of the RBa, polypeptlde 1s compared with those o1 the a, ~sotorms from rabbit cardiac and skeletal muscle. The amino acid residues are numbered at the right of the sequence for the RBa, protein. Amino acid identity 15 Indicated by a dot, and gaps are indicated by a dash. The putative membrane-spanning regions SI-S6 in each of the repeated domains I-IV arc’ indicated by bold lines and labeled.

rare form of splicing can be retained among related families of genes. Cytoplasmic Domain between Motifs I and II Two cDNA clones (RBII and RB520) were character-

ized and found to encode a different sequence in the intracellular region between motifs I and II. To determine whether additional sequences encoding this region were possible, the PCR was utilized to am-

Multiple

lsoforms

of a Rat Brain Calcium

Channel

41

9.6 7.5 -

8.6 - 6.5

4.3

Figure 5. Distribution

of RBa, in Rat Tissues

mRNAs from various rat tissues were analyzed by Northern blots as described in Experimental Procedures. A Pvul-Sphl fragment of RB9 was used as probe. The blot was washed two times for 15 min at room temperature in 2x SSC, 0.1% SDS and then two times inO.lx SSC,O.l% SDSat60°Cfor30min.Autoradiography was carried out at -80°C for 3 days with the exception of skeletal muscle, for which the exposure time was 20 hr. Brain’ is an autoradiograph of rat brain mRNA hybridized with PCR 1, an RBa,specific clone. The washing conditions were identical to those describedabove. Exposuretimewas2daysat-8OOCwith intensifying screens. The positions and sizes (kilobases) of the RNA marker are shown on the left side, and the calculated sizes of the transcript are indicated on the right side.

plify the variable region between these clones. As illustrated in Figure 2A, three clones of different sizes were amplified. The amino acid sequence of these different forms is presented in Figure 6A, along with the optimal alignment of these forms with the cardiac

A.

B.

Intracellular

53

of

Motif

Loop

Between

Motifs

I

and

sequence. RB17 was the longest amplified product; clones RB34 and RBII contain a 12 and 20 amino acid deletion, respectively, when compared with RB17. The amino acid sequence of RBII most closely resembles that of the heart isoform. In the skeletal isoform this region was shown to be less important than the link between motifs II and III in excitation-contraction coupling, although it was suggested that both the I-II and the ll-lll loops are critical and that the I-II loop may be more functionally interchangeable between the cardiac and skeletal al isoformsthan is the ll-lll loop (Tanabe et al., 1990). In the brain isoform it ispossiblethatthesethreeformsaltertheac:cessibility of the potential phosphorylation site at Ser-464, since this residue is only 17amino acids upstream from this variable region. Another interesting feature of these three isoforms is that the 22 amino acid insertion present in clones RB34and RB17contains7 basic residues. This positively charged region may interact with the acidic residues immediately upstream or the basic residues downstream of this variable region. Recently, the full-length sequence of the calcium channel from rabbit lung was publishecl. This sequence varied from the cardiac channel in four regions, apparently as a result of alternative splicing of a common transcript. It is interesting to note that an insertion of 25 amino acids was found inI this lung sequence in the region of the motif I-II linker immediately 5’of the variable region reported here. Although these regions showed no sequence homology, the fact that variability is observed in the same region of two different genes suggests the importance of this

II

IV

Figure 6. Alternative

Splicing

of RBa,

A model of the membrane topology of RBu, illustrates the areas of alternative splicing (bold line). (A) The intracellular loop between motifs I and II. The optimal amino acid alignment of the three clones encoding the cytoplasmic region between motifs I and II versus the cardiac sequence. (B) S3 of motif IV. Two forms of this brain protein encoding different sequences in the S3 of motif IV were found. The amino acid alignment of clones RB9 and RB48 is compared with the rat aorta counterparts, VSMu, and VSMa, ,, The boundries of the S3 of motif IV are indicated by bold line. (C) Carboxyl terminus. This is truncated compared with skeletal and heart a,.

Figure 7. PCR Analysis RBa,

Design

of the Carboxy-Terminal

End 01

primer kit (t’harmacla LKB Biotechnology, Inc., I+sc.ataway, Nli. and 0.5 x IO” dpmiml were added to the hybridization solution (50% deionized tormamide, 10% dextran sulfate, 6x SSPE, L s Denhardt’s solution, 0.7% SDS, 150 &ml denatured salmon sperm DNA). Hybridizations were carried out for 16 hr at 42°C The filters were washed two times for 15 min each at room tern perature In 6x SSC, 0.1% SDS, followed by two washes In 2, SSC, 0.1% SDS dt 60°C for 45 min. DNA from plaques that gave duplicate signals after three success1vc rounds of clonal purifica tion were isolated by a standard )i DNA liquid lysate procedural (Gubler and Hoffman, 1983)

The locations of primers RB3T, RBCT, and RB3U in relation to the 3’end of RBa, are indicated. Nucleotides 1500 and 2300 refer to RB9 and correspond to nucleotides 5142 and 5942, respectively, of the full-length product. The common region refers to that portionof RBalsimilartotheal subunitfrom heart.Theexpected sizes of the respective amplification products are indicated.

Characterization and Sequencing Positive clone\ were further confirmed by Southern blot and/ysis. cDNA Insert\ were subcloned into Ml3 mp18/19 or pBluc> script KS+ (Stratagene) by conventional technique\ (Maniatis et al., 1982). DNA sequencing was performed on both strands tor all reported clones using the dideoxy chdin termination method (Sanger et al., 1977) (T7- and deaza-T7 sequencing kits were purchased from Pharmacia LKB Biotechnology, Inc .i

region in determining the specific properties of these respective genes. The Carboxyl Terminus The a, subunit of the brain calcium channel cDNA is truncated compared with the skeletal and cardiac muscle a, subunits (311 and 482 amino acid differences, respectively). Interestingly, a native, truncated carboxy-terminal form of the a, subunit from skeletal muscle has been reported (De Jongh et al., 1989; Lai et al., 1990). Presumably this smaller form arises from a posttranslational processing event and may carry out functional roles different from those of the fulllength protein. Because the carboxyl termini of voltagedependent ion channels have been reported to have modulatory functions (VanDongen et al., 1990; Regulla et al., 1991), we verified the position of the stop codon in the brain a, subunit. Two different PCRs using different upstream primers amplified DNA fragments of the expected sizes (467 and 751 nucleotides) (Figure 2B; Figure 7). These PCR products were subcloned and sequenced and exhibited 100% homology with the 3’ RB9 clone. Therefore, the predicted truncated carboxyl terminus of RBa, is due to a shorter primary transcript.

Polymerase Chain Reaction To analyze the differences between clones encoding the cytfl plasmic region between motifs I and II, the PCR was utilized (Saiki et al., 1985). DNA was isolated trom the previously Deb scribed igtll rat brain library ustng a standard liquid lysate prc)~ cedure. The PCR was carried out in a volume of 100 ~1 containing 1 wg of DNA, 200 pmol of edch 30-mer oligonutleotide primer (RB + S-GAAACACTACCATCCCCACCAGTCACACCC-T and RB - Y-CTGCATCTCCTCCGATFGAACCGG-3’). 1 x Replinase but fer, 1 U of Prrtec t Match (Stratagene), 400 PM dATP, dCTP, dTTP. and dGTP (each,, and 2.5 LJ of Replinasr (Dupontl. The PCR wa\ overlayed with 100 PI of mlneral 011 and amplified under thr> following reactmn conditions: an initial denaturatlon at 94OC for 3 min, 40 cycles ot 94OC for 1 mln, 60°C for 30 $,72”C for 30 s, and a final extension at 72OC tor 10 mbn. The rcactlon product wd\ blunt-ended with 74 DNA polymerasr, klnased using 74 polv nucleotide klnase, dnd ligated Into theSmal siteof pBluescnpt KSt All reactions were run with the appropriate positive and negativcl controls. len pert ent of the reaction product was electrophoresed through an 81, nondenaturing polyac rylamide gel PCR analvbl\ was also used to analyze the truncated terms ot the carboxy-trrmlnal end ot RBu,. Batches ot rat brain mRNA differing from those used to construct the librartes (Isolated a, described below) served as a template. Both reverse transcription and PCRs were carried out in one \tep In a total volume ot 50 ~1. The reactIon mlxturr rontalned 2 pg ot mRNA. 400 PM dNTPs (each). L U of AMV reverse transcrlptasc. 1.25 U of Replinase (New England Nuclear), I U ot Perfect Matrh (Stratagemi. and 2.5 kg ot each primer. The folIowIng primer \equente\ were used. RB.37 5’.C;T<, ATGGGACACTCATC~CAATGCAACT-3’; RB3U S-ATCGGCCATCGTCAGCAACT-3’. RBCT 5’AACACCACGATCGCCCTACA-3’ is?< Figure 7). The reaction mixture wds overlayed with 100 PI ot mineral 011, and cDNA was synthesized In the first cycle at 42°C‘ for 40 min. After initial denaturation tor 10 min at 94OC, PCR products wereamplified in 30cycles, using an annealingtemperature60°C.The reaction wascompleted byafinal polymerization for 5 min at 72°C. The reaction products were subcloned a, described abovt.

Conclusion This study reports the complete sequence ot a novel isoform of the a, subunit of the voltage-dependent calcium channel. Additionally, we have characterized two variable regions of this protein, which lead to the potential generation of numerous subtypes of this isoform. Although the exact nature of these differences is currently not understood, future studies can now be undertaken to examine the specific functional consequences of these various isoforms. Experimental

Procedures

Screening of cDNA Libraries A rat hippocampal hgtll cDNA library pure hased tram Clonetech (Palo Alto, CA) was screened under the following conditions: 1 x IO6 hgtll recombinants were plated and transferred to Nytran filters (Schleicherand Schuell, Keene, NH) in duplicate. The cDNA probes were labeled with [“P]dCTP using a random

Isolation of RNA and Northern Blot Analysib Total RNA was Isolated from freshly excised rat tissues by the guanidinium Isothiocyanate-phenol procedure (Chomczynskl and Sacchi, 1987). Poly(A)+ RNA was isolated from total RNA bv two successive passes over an oligo(dT)-cellulose spin column (Pharmacia LKB Biotechnology, t nc.). Five micrograms of poly(A)’ RNA from various rat tissues was denatured, electrophoresed through a I% agarose, 0.8% formaldehyde gel (Maniatis et al.. 19821, and transferred to Nytran filters. The RNA was fixed bv baking the blots for 2 hr at 80°C under vacuum. The Northern blots were prehybridized for 3-6 hr and hybridized for 20 hl at 42’C in 50% formamide, 10% dextran sulfate, 6x SSPE, 2%

Multiple

lsoforms

of a Rat Brain

Calcium

Channel

43

Denhardt’s solution, 0.7% SDS, 150 mglml denatured salmon sperm DNA. ThecDNA probeswere labeled with [‘*P]dCTP using a random priming oligo labeling kit (Pharmacia LKB Biotechnology, Inc.), and 0.5 x lob dpmlml were added to the hybridization solution. The blotswerewashed twice for 15 min at room temperature in 2x SSC, 0.1% SDS and then twice for 30 min at 60°C in 0.1 x SSC, 0.1% SDS. Autoradiography was carried out at -80°C with two intensifying screens (Cronex). Sequence Analysis All nucleotide and amino acid analyses were carried out using the DNANALYZE program developed by Mr. Gregory Wernke of the University of Cincinnati, College of Medicine (Wernke and Thompson, 1989, Biophys. J., abstract).

partial amino acid sequence of a phosphorylation she of an inde pendent 8 subunit. Proc. Natl. Acad. Sci. USA 86, (8585-8589. De Jongh, K. S., Warner, C., and Catterall, W. A. (1990). Subunits of purified calcium channels, a2 and S are encoded by the same gene. J. Biol. Chem. 265, 14738-14741. Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., :Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W. J., Hui, A., Schwartz, A., and Harpold, M. M. (1988). Sequence and expression of mRNAs encoding the a, and a2 subunits of a DHPsensitive calcium channel. Science 247, 1661-1664. Fedulova, S. A., Kostyuk, P. C., and Veselovsky, N. S. (1985). Two types of calcium channels in the somatic membrane of new born rat dorsal root ganglion neurones. J. Physiol. 369, 431-446. Glossmann, H., and Striessnig, J. (1988). Characterization of the drug receptors of the purified skeletal muscle calcium channel. ISI Atlas Sci. Pharmacol. 2, 202-210.

Acknowledgments W e should like to acknowledge with thanks Mr. Greg Wernke for his expert assistance with the sequence analysis, Ms. Gwen Kraft and Mr. Donald Slish for help with the figures, and Rita Eveleigh and Gerald Chambers for typing the manuscript. We should also like to thank Dr. Kiyoshi Itagaki, Mr. Donald Slish, and Drs. Jeff Robbins, Gary Shull, Cyula Varadi, Dorothy Engle, and Walter Koch for helpful discussions and critical reviews of the manuscript. W e are grateful to Dr. Gary Shull for rat uterus and lung mRNAs, Dr. Koch for rat aorta mRNA, and Dr. Marilyn Kozak for confirmation of the unusual nature of the 5’ region of the cloned protein and advice regarding translational regulation. This study was supported, in part, by National Institutes of Health grants R37-HL43231-01 and POI-HL22619-12 (IC and Core 5) and a grant from Marion Merrell Dow Laboratories, Inc. Dr. Ronald J. Diebold is a special Postdoctoral Fellow of our Program in Excellence (NIH HL41496). The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank and is also available from the authors upon request. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.

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Added

Number

number

tar the bequencca,sr reported

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A. HUI and I’ I. lllinor

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to thl\ work