identification of a cardiac isoform of the murine calcium channel α1c (cav1.2-a) subunit and its...

Journal of Molecular and Cellular Cardiology 41 (2006) 115–125www.elsevier.com/locate/yjmcc

Original article

Identification of a cardiac isoform of the murine calcium channel α1C(Cav1.2-a) subunit and its preferential binding with the β2 subunit

Manabu Murakami a,⁎, Takayoshi Ohba b, Yoichiro Takahashi b, Hiroyuki Watanabe b,Ichiro Miyoshi c, Shinsuke Nakayama d, Kyoichi Ono a, Hiroshi Ito b, Toshihiko Iijima a

a Department of Pharmacology, Akita University School of Medicine, Akita 010-8543, Japanb Second Department of Internal Medicine, Akita University School of Medicine, Akita, Japan

c Center for Experimental Animal Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japand Department of Cell Physiology, Nagoya University Graduate School of Medicine, Tsuruma-cho, Shouwa, Nagoya, Japan

Received 30 January 2006; received in revised form 11 April 2006; accepted 1 May 2006

Abstract

We describe a cardiac muscle isoform of the voltage-dependent calcium channel α1 subunit, which corresponds to the rabbit ortholog of α1C-a(Cav1.2a). We also cloned smooth muscle isoforms α1C-b (Cav1.2b) and α1C-d (Cav1.2d). Differences among these three isoforms lie within theN-terminal region (exon 1A or 1B), the sixth transmembrane segment of domain I (exon 8A or 8B), and the use of exon 10, which forms theintracellular loop between transmembrane domains I and II. Two-hybrid analysis revealed interactions among the three α1 isoforms and βsubunits. In vitro overlay and immunoprecipitation analyses revealed preferential binding between α1C-a and β2, which is also expressed at ahigh level in the heart.© 2006 Elsevier Inc. All rights reserved.

Keywords: Voltage-dependent calcium channel; α1 subunit; Cardiac muscle; cDNA; Overlay; β subunit

1. Introduction

Voltage-dependent calcium channels (VDCCs) are the mainpathways by which calcium enters cells. VDCCs are multimericcomplexes of a main pore-forming α1 subunit and other auxi-liary subunits such as β, α2/δ, and γ. VDCCs play a crucial rolein the control of calcium-linked cellular functions and determinethe electrophysiological properties of cells. VDCCs are clas-sified pharmacologically into five groups (T, L, N, R, and P/Q),and 10 genes have been described for the pore-forming α1

subunits [1].L-type calcium channels are crucial for excitation–contrac-

tion coupling, which is the main pathway for calcium entry intoheart and smooth muscle tissue. L-type calcium channels are thetargets of calcium channel blockers such as dihydropyridine,which are used widely to treat hypertension and angina pectoris.Among the 10 α1 subunits that have been identified, four genes

⁎ Corresponding author. Tel./fax: +81 18 834 8930.E-mail address: [email protected] (M. Murakami).

0022-2828/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.yjmcc.2006.05.002

encode L-type calcium channels (Cav1.1, Cav1.2, Cav1.3, andCav1.4). Much attention has been paid to Cav1.2, which formsL-type calcium channels in cardiac and smooth muscle [1,2].Although a single gene encodes pore-forming α1 subunits inthese two clinically important tissues, the physiological pro-perties of L-type channels in cardiac tissue differ from those insmooth muscle [3]. For example, smooth muscle L-type chan-nels are much more sensitive to dihydropyridines, and this sen-sitivity depends on the membrane potential and other factorssuch as the channel assembly [4]. In addition, the Cav1.2 gene isresponsible for Timothy syndrome, which is a multiorgandisorder of autosomal-dominant heredity with a long QT syn-drome [5]. Although one cDNA isoform of Cav1.2 has beenreported to exist in neurons [6], whether other isoforms of theCav1.2 gene exist within the cardiovascular system has not beendetermined.

The channel pore-forming α1 subunit has four transmem-brane domains (I–IV). It is widely accepted that a high-affinityinteraction between α1 and β subunits occurs between twohighly conserved domains in each molecule. Specifically, these

mailto:[email protected]

http://dx.doi.org/10.1016/j.yjmcc.2006.05.002

116 M. Murakami et al. / Journal of Molecular and Cellular Cardiology 41 (2006) 115–125

subunits interact via the alpha-interaction domain (AID), whichis located between the first and second domains of the α1 sub-unit, and the beta-interaction domain (BID), which is located inthe secondary conserved domain of β subunit [7]. Additionalinteraction sites may also exist [8].

The purpose of this study was to identify novel isoforms ofCav1.2 in murine cardiac tissue. We identified three novel splicevariants of the Cav1.2 gene, which we designated Cav1.2-a,Cav1.2-b, and Cav1.2-d. Furthermore, we investigated the bindingaffinities of these novel isoforms of Cav1.2 for β subunits.

2. Materials and methods

2.1. RNA isolation and reverse transcriptase–polymerase chainreaction analysis

Total RNAwas isolated from the heart, brain, and aorta of aC57/BL6 mouse using the RNeasy Kit (Qiagen, Valencia, CA, USA).Reverse transcriptionwas carried out in a solution of 10 pmol oligo-dT primer, 1 μg RNA, 1× first strand cDNA buffer (Life Tech-nologies, Rockville, MD, USA), 10 mM dithiothreitol, 0.4 mMdNTPs, 40URNasin, and 200USuperscript II in a volume of 25μlat 42 °C for 45min. For the reverse transcriptase–polymerase chainreaction (RT-PCR), 2.0 μl of template was used. The full-lengthα1C-c specific sequence was amplified using primers H1s and (5′-ATGATTCGGGCCTTTGTTCAGCC-3′) and H2as (5′-CTA-CAGGTTGCTGACGTAGGACCT-3′), which correspond tothe murine Cav1.2 subunit sequences M1IRAFVQP8 and R2163

SYVSNL2169 (including the stop codon), respectively. To amplifythe full-length cDNAofα1C-c, we used primers H2as andC1s (5′-ATGGTCAATGAAAACACGAGGATG-3′), which correspondsto M1VNENTRM8. Splice-variant sequences (with/without exon10) were amplified using primers 5s and (5′-ACCATGGAGGG-CTGGACAGA-3′) and 4as (5′-AGACTCAGTCTCACTTGT-GGG-3′), which correspond to the murine Cav1.2 subunitsequences T393MEGWTD399 and G496APAGLHD503, respective-ly. The amplified sequences were subcloned into the pZero-2 plas-mid (Invitrogen, Carlsbad, CA, USA) and were sequenced. Thesequenced cDNA fragments were then inserted into DsRed-C1(Clontech, Palo Alto, CA, USA) or pTRG (Stratagene, La Jolla,CA, USA) plasmids for expression analysis.

β2 and β3 subunit-specific sequences were amplified byPCR (35 cycles) with the following primers: B2S (5′-CTAGA-GAACATGAGGCTACAG-3′) and B2A (5′-ACTGTTTG-CACTGGGCTTAGG-3′), corresponding to the sequences ofthe murine β2 subunit L131ENMRLQ137 and P198KPSANS204,and B3S (5′-CTCAAACAGGAACAGAAGGCC-3′) and B3A(5′-CATAGCCTTTCAGAGAGGGTC-3′), corresponding to thesequences of the murine β3 subunit L129KQEQKAR136 andP185SLKGYE191.

To obtain constructs for the overlay analysis, we used primer4as as well as the following primers:

7s (5′-AAAGAGAGGGAGAAAGCCAAA-3′;K440ERE KAK446);

14s (5′-GCCCGAGGAGATTTCCAGAA-3′;A446RGD FQK452);

13as (5′-TAGTTGCTGCTTCTCTCGAAG-3′;L453REKQ QL459);

12as (5′-GTAGCCTTTGAGATCTTCTTCTAG-3′;L459EED LKGY466);

11as (5′-TTCTGCCTGGGTGATCCAGTCC-3′;L467DWIT GAE474);

8as (5′-ATCATGCAAGCCCGCTGGAGC-3′;RGAPAG LH);

9as (5′-CCAAGCAAACTTCCCTTTC-3′;DGKKGKFAW); and

10as (5′-CATGCTCACATGGGTTTCTGTA-3′;STETH VSM).

2.2. BacterioMatch two-hybrid assay

Full-length sequences of murine β2 and β3 were cloned intothe pBT-bait plasmid (Stratagene), which contained λcI proteinto activate the λ operator. PCR-amplified fragments of α1C-a,α1C-b, and α1C-d (amplified using primers 5s and 4as) weresubcloned into the pTRG-target plasmid (Stratagene), whichcontained RNAP-α. These plasmids were used to transform thereporter strain (Stratagene), which was grown initially on agarplates that contained tetracycline, chloramphenicol, kanamycin,and carbenicillin (12.5, 34.0, 50.0, and 250.0 μg/ml, respec-tively; TCKC-agar plates). After this initial TCKC-agar plate-based screening, individual clones were further verified using asecondary marker, namely the β-galactosidase gene, on X-gal-containing (80 μg/ml) TCKC-agar plates [9].

2.3. Cell culture

Human embryonic kidney 293 (HEK-293) cells were cul-tured in Dulbecco's Modified Eagle's Medium supplementedwith 10% dialyzed fetal bovine serum. HEK-293 cells weretransfected with expression vectors that carried a portion of thecloned murine α1C sequence into the C-terminal region ofDsRed in the pDsRed-C1 vector (Clontech). This permittedimmunodetection and immunoprecipitation with anti-DsRedantibody (Clontech) after transient expression using the Lipo-fectamine Plus reagent (Invitrogen) [10].

2.4. Immunoprecipitation

Immunoprecipitation was performed using a protein G im-munoprecipitation kit (Sigma, St. Louis, MO, USA). The cellpellet was re-suspended in 1.0 ml of lysis buffer (20 mM sodiumphosphate, 150 mM sodium chloride, 10% glycerol, 1 mMethylenediaminetetraacetic acid, 0.5% Triton-X 100, pH 7.2)and complete TM protease inhibitor cocktail (Roche, Basel,Switzerland) at a concentration of ∼1 mg/ml. This suspensionwas set on ice for 1 h before being centrifuged at 10,000 × gat 4 °C for 15 min. The cleared lysate was incubated at 4 °Cfor 1 h with 2 μg of a polyclonal antibody directed againstthe DsRed-tagged protein, which was inserted at the N-terminus of each expression construct. Protein G sepharose(50 μl) was added to the samples, which were incubated for16 h at 4 °C. The immunoprecipitate was washed five times

117M. Murakami et al. / Journal of Molecular and Cellular Cardiology 41 (2006) 115–125

with 1 ml of immunoprecipitation buffer before being elutedwith 60 μl of Laemmli's buffer. The eluted products (15 μl)and an equal volume of whole-cell lysate were subjected to6% sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) and were then analyzed using a Western blot(ProBlot II AP; Promega, Madison, WI, USA) with anti-β2(Sigma) or anti-β3 (Alomone, Jerusalem, Israel) polyclonalantibodies. Densitometric results of three independent experi-ments were expressed as the mean ± SE.

2.5. In vitro overlay experiments for interaction between α1Cand β2

Human placental alkaline phosphatase (AP)-fused β2protein was synthesized as a fusion construction between asignal peptide and heat-stable AP under the control of theCytomegarovirus (CMV) promoter [11]. The murine β2 subunitwas fused to the N-terminus of AP in-frame with the Igκ-chainsignal peptide. This construct resulted in the secretion ofartificial AP-tagged β2 into the culture medium after expressionin HEK-293 cells. For transient expression, subconfluent HEK-293 cells in a 35-mm culture dish were transfected with 0.8 μgof the construct using Lipofectamine (Invitrogen). After 48 h,the medium was collected and the secreted fusion protein wasenriched with Amicon (Millipore, Bedford, MA, USA). Toestimate the concentration of the fusion protein, AP activity wasquantified by adding the substrate for this enzyme, ρ-nitrophenyl phosphate, which is converted by AP into a yellowproduct that can be measured at an absorbance wavelength of405 nm [11]. Because native AP has 15,000 unit/mg of enzymeactivity and a molecular weight of ∼67 kDa, the concentrationof the fusion protein (∼129 kDa) was estimated according toFlanagan's protocol. Transient expression of various α1Cconstructs was conducted as described above. After 48 h,samples of cell lysate were prepared in 1 ml of lysis buffer.

After separation by 6% SDS–PAGE, proteins were trans-ferred to polyvinylidene fluoride (PVDF) membranes. Themembranes were blocked in TBST (150 mM NaCl, 20 mMTris–HCl [pH 7.5], 0.05% Tween-20) with 0.1% bovine serumalbumin for 1 h, followed by incubation (16 h at 4 °C) in thepresence of 10 pM TBST containing complete TM proteaseinhibitor cocktail (Roche). The membranes were washed threetimes with TBS (150 mM NaCl, 20 mM Tris–HCl [pH 7.5])before AP activity was measured using the stabilized substratefor AP (Western Blue; Promega). Densitometric values of threeindependent experiments were expressed as the mean ± SE.

2.6. Quantitative analysis of binding between α1C-a andAP-fused β2

To assay specific AP-tagged β2 binding to α1C-a,subconfluent HEK-293 cells were transfected first with theDsRed-fused α1C-a construct for 48 h. Cells were thenwashed with HBHA buffer (Hanks' balanced salt solution with0.5 mg/ml bovine serum albumin, 0.1% NaN3, 20 mMHEPES, pH adjusted to 7.0). The plates were then incubatedfor 90 min at 4 °C with various concentrations (0.4–86.0 nM)

of AP-tagged β2 or corresponding concentrations of untaggedAP to estimate nonspecific AP activity (nonspecific binding).The cells were then rinsed seven times with HBHA bufferbefore being lysed in 50 μl of a solution of 1% Triton-X 100and 10 mM Tris–HCl (pH 8.0) [11]. The lysate was heated to65 °C for 10 min to inactivate cellular phosphatases. Theremaining heat-stable AP activity was evaluated by adding thesubstrate ρ-nitrophenyl phosphate, as described above.

2.7. Deletion analysis of isoforms

Serial deletion constructs of exon 10, which distinguishesα1C-b from α1C-d, were prepared with PCR using primers 7s,10as, 9as, and 8as. The aforementioned DsRed-fused α1C-bconstruct, which was prepared with primers 7s and 4as, wasused as a template.

To analyze the interaction between α1C-a and β2, serialdeletion constructs of exon 8A, which are used specifically inα1C-a and α1C-c, were prepared using primers 7s, 14s, 13as,12as, and 11as. The DsRed-fused α1C-a construct, which wasprepared using primers 7s and 4as, was used as a template.

Each PCR-amplified fragment was subcloned into the C-terminal region of the DsRed expression vector. Each DNAexpression construct was expressed independently and tran-siently in HEK-293 cells. The DsRed-tagged proteins wereseparated using SDS-PAGE before being transferred to PVDFmembranes. Overlay analysis was carried out using therecombinant AP-fused β2 subunit.

2.8. Mutagenesis in exon 8

We used primer 4as together with a relatively long de-generate primer called Ex8MUT (5′-RTGMAWGACGC-TRTGGGCWRKGASTKGCCCTGGRTGTATTTTGT-CASTCTGRTCATCWTAGGGTCCTTT-3′) to amplify allpossible amino acids encoded by exon 8A (specific to α1C-a)and 8B (specific to α1C-b and -d). We used α1C-a as thetemplate. The PCR-amplified DNA fragments were subclonedinto the C-terminal region of the DsRed expression vector and48 independent expression constructs were prepared. Theconstructs were purified and expressed in HEK-293 cells beforebeing subjected to an overlay analysis as described above.

2.9. Statistics

Differences were evaluated using unpaired t tests, unlessstated otherwise. The level of statistical significance was P<0.05.

3. Results

3.1. Identification of murine cardiac α1C transcripts

Our cloning strategy was based on a detailed search forhomologs of full-length rabbit α1C-a (accession numberX15539) andα1C-b (accession number X55763) in an expressedsequence tag database using the program BLASTN (NationalCenter for Biotechnology Information; http://www.ncbi.nlm.nih.

http://dx.doi.org/doi:10.1016/j.yjmcc.2006.05.002


gov/blast/). Five clones (accession numbers AK052162,U17869, L01776, NM-009781, and BB473606) were identifiedas murine orthologs of α1C (Cav1.2), but none of these clonesappeared to be the full-length sequence. All clones sharedoverlapping cDNA sequences that were found to be partialsequences of a single gene. The sequence of one clone(AK052162) resembled the N-terminal sequence of rabbitα1C-a; therefore, we amplified the full-length sequence of ourcDNA using RT-PCR primers that were based on the partialsequence of AK052162.

Fig. 1. Detection of novel isoforms of murine Cav1.2. (A) Detection of murine transcr6510-bp length product equivalent to the full-length form of cardiac Cav1.2a (α1C-a)text for primer sequences). (B) RT-PCR analysis of exon use in different tissues from aAo, aorta; He, heart; Br, brain; In, intestine; Co, colon; Nc, negative control (no cDNAnot contain exon 10. (C) Identification of β2 and β3-specific transcripts in the muricontrol (no cDNA). Primer sets used for each PCR amplification are indicated above.use of exons 1 (A or B), 8 (A or B), and 10. The positions of the primers (5s and 4as) adomain I, the alpha-interaction domain (AID), and the intracellular loop are also indicCav1.2a (a), Cav1.2b (b), Cav1.2d (d), and neuronal Cav1.2c (c) isoforms. Alignmentcalculated relative to primer 5s, are numbered in the right margin. Sequences are predomains are underlined in black. Asterisks denote alignment of three identical residueamino acids within the AID (open circles) are indicated. Phenylalanine (421F) is al

Murine heart total RNA was reverse-transcribed and am-plified by PCR using the specific primers H1s and H2as (Fig.1A). No PCR products were amplified in the negative controlreaction, which contained the mixture used for PCR amplifi-cation without the product of the reverse transcription reaction.The PCR-amplified fragment was subcloned and sequenced.The 6510-bp product was found to be an ortholog of rabbitα1C-a (2). Furthermore, a search of a genomic DNA databaserevealed two types of splice variant: one exhibited N-terminalsplice variation (exon 1B) and the other was the result of

ipts of Cav1.2a by reverse transcriptase–polymerase chain reaction (RT-PCR). Awas amplified from mouse heart cDNA using H1s and H2as as primers (see maindult mice (16 weeks old). RT-PCR primers 5s and 4as were used. Abbreviations:). The long fragment (414 bp) contained exon 10; the short fragment (339 bp) didne heart (He) and aorta (Ao). Abbreviations: He, heart; Ao, aorta; Nc, negative(D) Exon use in α1C-a, α1C-b, α1C-d, and α1C-c. Differences depended on there indicated above the corresponding exons. The last transmembrane segment inated. (E) Comparison of the amino acid sequences and structural alignment of thewas performed using the program ClustalX (ver. 1.8). The amino acid positions,sented in the single-letter amino acid code. Amino acids in the transmembranes. The dashes represent gaps in the sequence. The AID (box) and three importantso shown (closed circle).



differential expression of exon 10; specifically, exon 10 was nottranscribed in α1C-a or α1C-c, which resulted in differentamino acid sequences for these two isoforms. The GenBankaccession numbers are AB259049, AB259050, and AB259051,α1Ca, α1Cb, and α1Cd, respectively.

3.2. PCR amplification of murine smooth muscle α1C

To analyze tissue-specific transcription of different α1Cisoforms in smooth muscle, we constructed a pair of PCRprimers that allowed us to distinguish between isoforms with/without exon 10. These primers, namely 5s (derived form exon7) and 4as (derived from exon 11), corresponded to the aminoacid sequences of the extracellular loop between S5 and S6 ofthe first transmembrane domain (domain I) and the intracellularloop between domains I and II, respectively.

Fig. 1B shows a representative result of the RT-PCR analysis.The aforementioned pair of primers amplified a single band(339 bp) from heart (He) and two bands (339 and 414 bp) fromaorta (Ao). The longer fragment of the two PCR-amplified bandsfrom aorta (414 bp) was the major product and corresponded toα1C-b, while the shorter band (339 bp) was relatively weak. Theshorter isoform lacked the complete sequence of exon 10 andwas named α1C-d, which will be discussed later. The same pairof PCR primers that amplified a single short band from heart(He) amplified two distinct bands from brain (Br), intestine (In),and colon (No). Sequence analysis revealed that the PCRproducts from heart and brain both contained exon 8A, whichcorresponds to rabbit α1C-a and α1C-c. In contrast, the PCRproducts from the other tissues that contained smooth musclecontained the sequence of exon 8B. The longer of the two bandsthat contained exon 10 corresponded to α1C-b while the shorterband corresponded to α1C-d.

Comparative RT-PCR with RNA products derived fromheart (He) and aorta (Ao) tissue were analyzed using the β2-specific primers B2S and B2A, and β3-specific primers B3Sand B3A (Fig. 1C). RT-PCR analysis of the heart and aortatissue showed β2- and β3-specific bands of 222 and 187 bp,respectively. Our RT-PCR analysis suggests that β2 is probablythe major β subunit in heart tissue, as β3-specific primershowed only a faint band in this tissue, although PCRamplification is influenced by primer sequences and otherexperimental conditions. However, both primer pairs showedfair expression of β2 and β3 subunits in the aorta tissue,suggesting the expression of both subunits. We also performedthe experiment with β1- and β4-specific primers, which did notyield any PCR products (data not shown).

3.3. Structure of the murine α1C (Cav1.2) subunit gene

We searched a genomic DNA database using the rabbit α1C-a and α1C-b cDNA sequences and identified a contiguous clonein Chr6F1 (accession number NT_094510.1) and 46 differentmatches with two alternative exons that were identical to theDNA sequence of the murine α1C subunit. Two exons (1B and10) were not detected because the sequence of exon 1B issubstantially different from that of the rabbit ortholog while the

sequence of exon 10 was too short to be detected. Exon 1B wasfound using in silico analysis of the murine α1C-c sequences(accession numbers L01776, AY728090, and NM_009781).Detailed sequence analysis of the exonic and most of theintronic regions of the identified clones indicated that the codingregion of the gene extended over ∼602 kb, which is signifi-cantly longer than the estimated coding region of the humanα1C subunit (∼150 kb) reported by Soldatov [12]. The fourthintron was longest ( >272 kb) and was located between the thirdand fourth exons. The α1C subunit gene contained 48 translatedexons, two of which formed alternative exons, and existed as asingle-copy locus in the haploid mouse genome.

3.4. Exon use in the α1C gene

Previous reports revealed that α1C subunit transcripts arespliced preferentially at the N-terminal region and the internalloop between domains I and II [2,13,14]. Fig. 1D illustrates thecomprehensive exon use in the α1C-a, α1C-b, and α1C-d splicevariants based on our sequence analysis of identified RT-PCRclones. The differences in exon use among these splice variantsdepend on the use of exons 1 (A or B), 8 (A or B), and 10 (Fig.1E). The novel cardiac isoform of α1C contained exon 1A andthe exons that followed exon 8A, while exon 10 was skipped.The skipping of exon 10 resulted in a change in the amino acidsequence from valine (α1C-b) to methionine (α1C-a, -c, and-d). The smooth muscle isoform contained exon 1B and theexons that followed exon 8B; some isoforms did (α1C-b) whileothers did not (α1C-d) contain exon 10. The neuronal isoformof α1C contained exon 1B and the exons that followed exon 8A,but did not contain exon 10.

3.5. Interaction between α1C isoforms and β subunits

The interaction between domains I and II and β subunits hasbeen characterized extensively and has been ascribed to a singleAID [15–18]. Although De Waard et al. [16] publishedevidence of a possible second site in the AID that binds β2,this has not been demonstrated. To investigate whether therewas binding between the novel isoforms of the murine α1Csubunit and β subunits, we used a two-hybrid analysis. To ourknowledge, this approach has not been used previously toexamine interactions between these molecules. Figs. 2A and Billustrate the plasmid constructs used for the two-hybridanalysis. Partial sequences of the isoforms of the murine α1Csubunit were fused to the C-terminal region of RNAP-α protein(Fig. 2A), while β2 (i) or β3 (ii) subunits were subcloned in-frame into the C-terminal region of λcI protein (Fig. 2B). Theresults of the two-hybrid analysis revealed that the α1Cisoforms interact with β2 and β3 subunits (Fig. 2C).

3.6. Preferential interaction between α1C-a and β2 subunits

It has been established previously that α1C interacts with β2subunits, but whether the different splice variants of α1Cexhibit different affinities for β2 is not known. To furthercharacterize the binding between α1C and the β subunits, we

Fig. 3. Coimmunoprecipitation analysis of interactions between α1C isoformsand β subunits in human embryonic kidney 293 (HEK-293) cells. (A)Expression constructs of α1C (i–iii correspond to α1C-a, α1C-b, and α1C-d,respectively) and exon use in each construct (right). HEK-293 cells were co-transfected with the DsRed-tagged α1C isoforms and β2 or β3 subunits.Immunoblots of cell lysates (left, i–iii) and immunoprecipitation productsobtained using an anti-β2 antibody (Biv), anti-β3 antibody (Bv), and anti-DsRed antibody (Bvi) are presented. The α1C isoforms are indicated as a(α1C-a), b (α1C-b), and d (α1C-d). Lanes: 1, β2; 2, co-expression of β2 andβ3; and 3, β3. The β2 subunit probe revealed weak binding with α1C-b(panel iv, b, lanes 1 and 2, asterisk) and marginal binding with α1C-d (paneliv, d, lanes 1 and 2, arrow), while the β3 subunit did not exhibit anydifference in binding (v, lanes 2 and 3). The anti-DsRed antibody revealedequal loading of each α1C isoform in the cell lysates (iii) and the immuno-precipitation products (iv).

Fig. 2. Two-hybrid analysis of interactions between the α1C isoforms and βsubunits. (A) DNA constructs of α1C isoforms. PCR-amplified α1C isoformswere subcloned into pTRG (‘target’) and fused to the C-terminal region ofRNAP-α under the control of the lac-UV5 promoter. (B) DNA constructs of β2and β3 subunits for the bacterial two-hybrid analysis. The full-length clone ofthe β2 or the β3 subunit was subcloned into pBT (“bait”). These constructs werefused to the C-terminal region of λ-C1 under the control of the lac-UV5promoter. (C) Screening of interacting proteins with the bacterial two-hybridsystem. After first screening with tetracycline, chloramphenicol, kanamycin,and carbenicillin, positive clones were plated onto X-gal-containing (80 μg/ml)indicator plates. Colonies that were dark reflected the induction of the β-galactosidase gene. The names of the “target” α1C isoforms (a, b, and d) appearabove, while the names of the “bait” (β2, β3, and negative control, NC) appearto the left.


used an immunoprecipitation analysis (Fig. 3). To express thevarious α1C isoforms (α1C-a, -b, and -d) in HEK-293 cells,partial sequences that were PCR-amplified using primers 5s and4as were subcloned into the C-terminal region of DsRed proteinthat was under the control of the CMV promoter (Fig. 3Ai–iii).The cDNAs of β2 (lane 1) and β3 (lane 3) were co-transfectedinto HEK-293 cells together with each of the various α1Cisoforms. To detect differences in binding affinity, we co-transfected the same amount of β2 and β3 (1:1 ratio) and eachα1C isoform (lane 2).

As shown in Fig. 3B, Western blot analysis of the cell lysaterevealed that β2 (i), β3 (ii), and DsRed (iii) were expressed inthe transfected cells. The amount of protein expressed dependedon the amount of DNA that was used for the transfection.Immunoprecipitation analysis with an anti-DsRed antibody andimmunodetection with an anti-β2 antibody revealed that therewas strong binding between α1C-a and β2 (Fig. 3Biv, a, lane 1),moderate binding between α1C-b and β2 (39.3 ± 8.8% of thebinding intensity of α1C-a and β2; P < 0.05, Fig. 3Biv, b, lane1, asterisk), and marginal binding between α1C-d and β2 (6.5 ±0.7% of the binding intensity of α1C-a and β2, P < 0.05; Fig.

3Biv, d, lane 1, arrow). Analysis of competitive co-expressionof β2, β3, and the various α1C isoforms revealed similardifferences in the interaction between the α1C isoforms and β2(46.7 ± 3.5, 14.3 ± 5.4, 6.4 ± 1.8% of the binding intensity ofα1C-a and β2 (Fig. 3Biva, lane 1), α1C-a, b, d, respectively,Fig. 3Biv, a, b, and d, lane 2). In contrast, there were nodifferences in binding between each of the α1C isoforms and β3(100.0 ± 9.2, 84.0 ± 6.7, 97.6 ± 2.9% of the binding intensity ofα1C-a and β3 (Fig. 3Bva); α1C-a, b, d, respectively, Fig. 3Bv,lane 3). The amount of β3 that was immunoprecipitateddepended on the amount of DNA used for the transfection(lanes 2 and 3). The anti-DsRed antibody revealed thatimmunoprecipitation produced an equal amount of DsRe-d–α1C fusion protein for each of the isoforms (100.0 ± 7.2,105.4 ± 9.6, 106.4 ± 12.5% of α1C-a (Fig. 3Bvia, lane 1); α1C-a, b, d, respectively, Fig. 3Bvi). Collectively, these resultssuggest that the β2 subunit and α1C-a interacted preferentially.


3.7. Overlay analysis with AP-labeled β subunits

To further characterize the interaction between α1C and β2,we used an in vitro overlay assay with an AP-labeled β2 subunit(Fig. 4A). The various α1C constructs that were used for theimmunoprecipitation analysis (see above) were expressed tran-siently in HEK-293 cells. After 48 h, the proteins in the celllysates were separated using SDS-PAGE, transferred to PVDFmembranes, and probed with an anti-Myc antibody that re-cognized the C-terminus of the AP-tagged β2 subunit (Fig. 4B).A band that corresponded to AP-tagged β2 (129 kDa; Fig. 4Bi,lane 1) was revealed by the anti-Myc antibody. As a control, AP

Fig. 4. Overlay analysis and in vitro binding assay of the β2 subunit. (A)Expression construct of the β2 subunit. A start codon (ATG) was inserted at theN-terminal region of the Igκ-chain signal peptide. The full-length clone of theβ2 subunit was inserted into the N-terminus of alkaline phosphatase (AP) tocreate a signal peptide–β2–AP fusion protein. A Myc-tag was inserted at the C-terminus of AP. (B) Immunoprecipitation analysis of the recombinant β2–APprobe. After transient expression in HEK-293 cells, cell culture medium wascollected and incubated with anti-Myc antibody. The immunoprecipitationproducts were separated using sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and were transferred to a polyvinylidene fluoride(PVDF) membrane. The anti-Myc antibody revealed a single band thatcorresponded to the β2–AP fusion protein (lane 1, 129 kDa). Lane 2 containedthe immunoprecipitation products corresponding to the positive control APexpression construct. The anti-β2 antibody reacted only with the fusion proteinconstruct (β2–AP). (C) Overlay analysis with the β2–AP probe. Cell lysates ofHEK-293 cells that were transfected with DsRed-tagged α1C were separatedusing SDS-PAGE and transferred to a PVDF membrane. Overlay analysis withthe β2–AP probe revealed isoform-dependent signals. Lanes: a, α1C-a; b, α1C-b; and d, α1C-d (i). Anti-DsRed revealed fairly equal amounts of DsRed-α1Cfusion protein (ii). (D) Representative binding curves for the DsRed–α1C-afusion protein and various concentrations of the β2–AP probe (i). Scatchardtransformation of equilibrium binding (ii). Scatchard analysis revealed a high-affinity site (Kd = 8.5 nM, Bmax = 0.32 fmol/mg) and a low-affinity site (Kd =134 nM, Bmax = 0.79 fmol/mg).

with the Myc epitope was transfected and immunoprecipitated(61 kDa, lane 2). Anti-β2 antibody confirmed the expressionand specificity of the secreted AP-tagged β2 (Fig. 4Bii, lane 1),while the AP with the Myc epitope did not produce any signal(lane 2).

Interactions between each of the α1C isoforms and β2 weredetected by assaying the relative activity of AP that wastagged to the C-terminal sequence of β2 (Fig. 4Ci). The resultscorresponded to those of the immunoprecipitation analysis (seeFig. 3B). Specifically, the interaction between α1C-a and β2was strong (Fig. 4Ci, lane a). The interaction between α1C-band β2 was moderate (17.9 ± 2.1% of the interaction of α1C-aand β2, P < 0.05, lane b), while α1C-d interacted weakly withβ2 (3.4 ± 1.7% of interaction of α1C-a and β2, P < 0.05, laned). The expression of the DsRed tag was confirmed with ananti-DsRed antibody, which revealed that similar amounts ofthe fusion proteins were loaded (100.0 ± 3.9, 104.2 ± 9.6,121.6 ± 5.1% of the DsRed-intensity of α1C-a; α1C-a, b, d,respectively, Fig. 4Cii).

3.8. Binding analysis

To further investigate the characteristics of binding betweenα1C-a and β2, we carried out a saturation binding analysis inwhich α1C-a transfected HEK-293 cells were incubated withvarious amounts of AP-tagged β2 protein. Representativeresults of this analysis are presented in Fig. 4D. The bindingbetween transiently expressed α1C-a and AP-tagged β2 couldbe saturated with sufficiently high concentrations of AP-taggedβ2, although the relatively high background signal at such highconcentrations made further Scatchard analysis difficult (Fig.4Dii). The negative control was evaluated with correspondingconcentrations of AP, and specific binding at each concentra-tion was calculated by subtracting the remaining AP activity.The Scatchard analysis revealed two different dissociationconstants for the interaction between α1C-a and β2. Specif-ically, there appeared to be a high- and a low-affinity site with aKd value of 8.5 ± 2.5 nM (Bmax = 0.32 ± 0.08 fmol/mgprotein; n = 4) and 134 ± 28.5 nM (Bmax = 0.79 ± 0.21 fmol/mg protein), respectively. These observations concur with theresults of a previous binding analysis of 35S-labeled β2, whichrevealed a high- and low-affinity binding site with a Kd valueof 3.5 and 36.0 nM, respectively [16].

3.9. Deletion analysis

It is very likely that the AID of the α1C subunit has twobinding sites for β2, although the low-affinity site has not beenidentified [16]. The consensus sequence of the AID variesslightly among different studies [16,19]. The original AID isQQ-E–L-GY-WI—E [15], while Marquart and Flockerzireported that QQ-E–L-GY-WI is the minimal requirement foroverlay detection [19]. To characterize differences in the bindingcharacteristics of the different α1C isoforms and β2, weprepared deletion mutants of each of the α1C isoforms.

As stated above, we found that α1C-b and α1C-d hadmarkedly lower binding affinity for β2 compared to the binding


between α1C-a and β2 (see Figs. 3 and 4). To identify whichamino acids produced these differences in binding affinity,serial deletion constructs of exon 10 (which is downstream ofthe AID and distinguishes the cardiac and smooth muscleisoforms of α1C) were prepared for an overlay analysis withAP-tagged β2 (Fig. 5A). Deletion of the C-terminal portion ofexon 10 (PTSETES) had no effect on binding (105.9 ± 13.5% ofthe interaction of α1C-b and β2, P > 0.05, lane 2), but deletionof additional amino acids (SHSTETHVSM) caused a majorreduction in binding (41.2 ± 6.0% of the interaction of α1C-band β2, P < 0.05, lane 3). An additional deletion (DQKK-GKFAWFSH) did not further reduce binding (45.0 ± 4.7% ofthe interaction of α1C-b and β2, P > 0.05, lane 4). Overlayanalysis with AP-tagged β2 revealed that the C-terminalsequence of exon 10 was important for binding in α1C-b(Fig. 5A, lanes 3 and 4).

De Waard et al. [20] reported previously that three aminoacids within the AID, namely tyrosine (Y), tryptophan (W), andisoleucine (I), were important for binding the β subunit. Theseauthors found that mutation of each of these amino acids greatlyreduced binding between α1A and β1 subunits. In addition,they reported a possible secondary binding site. In the present

Fig. 5. Deletion analysis of α1C isoforms. (A) Deletion analysis of the smoothmuscle isoform, α1C-b. Amino acid sequences of α1C-b constructs in Ds-Red–α1C-b fusion protein. The AID and exon 10 are shown. Arrows indicatecorresponding amino acids and directions of each primer (7s, 10as, 9as, 8as, and4as). Overlay results for each construct (lane 1, 7s-4as; lane 2, 7s-8as; lane 3, 7s-9as; lane 4, 7s-10as). (B) Deletion analysis of the cardiac isoform, α1C-a. Aminoacid sequences of α1C-a constructs in DsRed–α1C-a fusion protein. The AID isindicated. Arrows indicate corresponding amino acids and directions of eachprimer (7s, 14s, 13as, 12as, and 11as) (i). Overlay results for constructs with the7s primer (lane 1, 7s-11as; lane 2, 7s-12as; and lane 3, 7s-13as) (ii). Overlayresults for constructs with the 14s primer (lane 1, 14s-11as; lane 2, 14s-12as; andlane 3, 14s-13as) (iii).

Fig. 6. Overlay analysis of mutations in exon 8. (A) Representative results of theoverlay for DsRed–α1C-a fusion constructs and the β2–AP probe (i) andcorresponding Western blot analysis with anti-DsRed antibody (ii). Lanes: 1,α1C-a; 2, clone #6; 3, clone #10; 3, clone #14; and 4, α1C-d. The amino acidsequences of the corresponding clones are listed below. (B) Deduced amino acidsequences of α1C-a (H, amino acids 402–425 of α1C-a), α1C-b/d (S), andmutants with low signals (1–16). Amino acids specific to α1C-a are underlined.Amino acids specific to α1C-b/d specific are in italics. Note that there was nophenylalanine (421F) in the analyzed clones (asterisk).

study, we prepared deletion mutants for various regions of theAID. Constructs were prepared with α1C-a as the templateDNA. Serial deletion constructs of the AID in exon 9 wereprepared (Fig. 5Bi). The construct that contained the full-lengthAID (QQLEEDLKGYLDWITGAE) exhibited strong binding(Fig. 5Bii, lane 1). The construct that contained the middleportion of the AID (QQLEEDLKGY) and a deletion of the C-terminal region (LDWITGAE; W and I deleted) exhibitedreduced binding (44.4 ± 3.1% of the full-length construct, P <0.05, Fig. 5Bii, lane 2). The construct that contained only thefirst two glutamines of the AID (QQ; Y, W, and I deleted)exhibited marginal binding (3.4±0.6% of the full-lengthconstruct, P < 0.05, Fig. 5Bii, lane 3). Therefore, the inclusionof the entire AID (Fig. 5B, lane 1), especially the middle portionof the AID (lane 2), was required for binding, which concurswith the results of previous studies [19,20]. The observation thatthe middle portion of the AID (QQLEEDLKGY) could stillsupport binding (albeit at a reduced level) suggested that theseamino acids might form the low-affinity binding site (Fig. 5Bii,lane 2). An even shorter construct with a deletion of the N-terminal portion (KEREKAKA) in exon 9 exhibited fairly low


binding affinity for β2 (27.4 ± 3.4*% of the interaction of α1C-a and β2, *P < 0.05, Fig. 5Biii, lane 1), while further deletion ofthe AID sequence produced only marginal binding with the β2subunit (11.5 ± 2.8* and 2.1 ± 1.1*% of the interaction of α1C-aand β2, *P < 0.05, Fig. 5Biii, lanes 2 and 3, respectively).

To analyze isoform-dependent differences in binding affinityin more detail, mutations were introduced in exon 8A using adegenerate primer (Ex8MUT) that covered all possible aminoacid combinations of α1C-a (exon 8A) and α1C-b (exon 8B).We randomly selected 48 independent constructs that had beeninserted into the C-terminus of DsRed. The purified DNAconstructs were expressed transiently in HEK-293 cells. Theexpressed proteins were separated by SDS-PAGE and trans-ferred to PVDF membranes for the overlay analysis. Fig. 6Ashows representative results of this overlay analysis. Ascontrols, α1C-a (positive control, lane 1) and α1C-d (negativecontrol, lane 5) were used (Fig. 6Ai). The signal intensities weredifferent, and we selected 18 clones with low affinity (e.g.,clone #6, lane 2; clone #10, lane 3; clone #14, lane 4).Densitometric analysis reveled a significantly reduced bindingintensity of each clone (23.3 ± 4.6*%, 9.8 ± 1.2*%, 1.1 ± 0.6*%, and 2.6* ± 1.6% of α1C-a, clone #6, #10, #14, and α1C-b,respectively, *P < 0.05 vs. α1C-a). Ponceau-S stainingconfirmed fairly equal amounts of the expressed proteins(85.9 ± 4.8%, 95.6 ± 4.8%, 95.8 ± 5.3, and 74.6 ± 7.4% of α1C-a, clone #6, #10, #14, and α1C-b, respectively, Fig. 6Aii). Eachclone was sequenced and the corresponding amino acidsequences of exon 8 are shown in Fig. 6B. Amino acidsequences of low-affinity clones were an almost randomcombination of the amino acid sequences of α1C-a and α1C-d, except for isoleucine in the S6 transmembrane segment(asterisk, italic) and leucine, which was encoded by thedegenerate PCR primer. Collectively, our results emphasizethe importance of phenylalanine (421F) in exon 8A forpreferential binding between α1C and β2.

4. Discussion

In the present study, we cloned a novel cardiac isoform of thepore-forming α1C gene, which corresponds to rabbit α1C-a. Inaddition, we discovered a novel splice variant of the murineα1C gene, namely α1C-d, which lacks exon 10 and is expressedin smooth muscle. Biochemical analyses revealed preferentialinteraction between α1C-a and the β2 subunit.

We initially analyzed the protein–protein interactionbetween α1C and β subunits using the Escherichia coli-based two-hybrid system, BacterioMatch. This is a fast andefficient method of detecting protein–protein interactions invitro. Because the reporter strain has a background level ofampicillin resistance due to basal transcription of the reportercassette, we used carbenicillin instead of ampicillin. Adisadvantage of the two-hybrid method is that binding affinitycannot be analyzed in detail. Nevertheless, this method isrelatively simple and sensitive. We also examined a two-hybrid epitope expression library of α1C cDNA, but failed toidentify any other binding sites in other regions of α1C (datanot shown).

To analyze binding in detail, we used immunoprecipitationwith the DsRed tag. We failed to observe subcellularlocalization of the DsRed-tagged proteins (data not shown)likely because the intensity of the DsRed (red) fluorescence isrelatively low and/or our fluorescence microscopy system isrelatively insensitive. Nevertheless, the immunoprecipitationanalysis revealed that the various isoforms of α1C exhibitdifferent binding affinities. The β2 subunit, which is probablythe major component of cardiac calcium channels, preferentiallybound α1C-a (Fig. 3Bv). De Waard et al. [20] reportedpreviously that the β2 subunit has two binding sites, specificallya high- and a low-affinity site (Kd = 3.5 and 36.0 nM,respectively), while the Kd of the β3 subunit is 55.1 nM.Although β3 subunits are expressed in cardiac muscle, theaforementioned binding affinities lead us to speculate that α1C-a preferentially forms channels in cardiac muscle by binding β2subunits rather than β3. These different binding affinities areprobably physiologically important because the properties ofchannels depend on the type of β subunits that compose thechannel [21,22]. For example, channels that contain the β2subunit have a much slower rate of inactivation than channelsthat contain the β3 subunit. The slower rate of inactivation ofβ2-containing channels might be advantageous for somephysiological processes, such as contraction of cardiac muscle,because cardiac muscles require a prolonged influx of calciumto circulate blood effectively.

To further analyze the interaction between α1C and βsubunits, we used AP-fused protein as a probe. Compared to theuse of radioactive 35S- or 125I-labeled ligands, the AP fusionmethod has several advantages. First, no radioactive materialsare needed; second, a labeling reaction is not required; third,ligand purification is minimal; and finally, the sensitivity of themethod is similar to the radioactivity-based method [11]. TheIgκ-chain signal peptide at the beginning of the AP-fusionprotein construct directs the secretion of the fusion protein intothe culture medium after the construct is transfected into HEK-293 cells. HEK-293 cells contain the SV40 large T antigen,which allows the replication of plasmids that contain the SV40origin of replication; this results in a high level of expression ofthe desired protein. This strategy has several advantages overconventional Western blot analysis, including the fact that site-directed antibodies are not required, the produced fusion proteinis stable for a long period.

To determine why the binding of β subunits by the smoothmuscle α1C isoforms (α1C-b and α1C-d) is significantlyweaker than that of α1C-a, we conducted a deletion muta-genesis analysis of α1C-b using an overlay assay (Fig. 5A). Ourfindings suggested that the middle portion of the codingsequence of exon 10, which is absent from α1C-d, influencedbinding affinity. In the overlay assay, we also attempted toidentify the second (low-affinity) binding site in α1C-a using adeletion analysis of exon 9. Our results showed that constructsthat contained only a portion of the AID (specifically,QQLEEDLKGY) could still bind the β2 subunit; therefore,this region likely corresponds to the lower affinity binding site.We also examined shorter peptide constructs, but the bindingaffinity of these constructs was too weak to be analyzed further.


A previous study demonstrated that three amino acids withinthe AID, namely Y, W, and I, are essential for the interactionbetween α1C and the β2 subunit [20]. Among these threeamino acids, tryptophan has been reported by others to beimportant for channel function [23]. Our results suggest asecond interaction site in addition to the aforementioned sitewithin the AID (Fig. 5Bii). The interaction between α1C-a andβ2 was partly dependent on the length of the AID bindingdomain, as indicated by the faint bands obtained in the overlayanalysis of the short construct (Fig. 5Biii). Mutagenesissuggested that a phenylalanine (421F) within exon 8A isimportant for binding; this amino acid is located within the I S6transmembrane segment 37 amino acids from the N-terminus ofAID. The mechanism that underlies binding is not yet clear, butthe results of our study suggest that exon 8A preferentiallybinds the β2 subunit in the heart. To our knowledge, onlyCav1.2 exhibits such a variety of alternative splicing of exons(exon 8A and 8B), and both of these exons encode a conservedtransmembrane segment (domain I S6). Further study is neededto elucidate the physiological importance of these exons.

Interestingly, mutation at the initial portion of the intracel-lular domain that follows the first transmembrane domain (I S6)causes Timothy syndrome [5]. Although the exact transmem-brane segment is difficult to predict, it is possible that theresponsible amino acid (R instead of G) is included in thetransmembrane segment (S6). Nevertheless, this portion ispathophysiologically important because Timothy syndrome is again-of-function mutation that results in marginal inactivationof calcium currents. Therefore, the mutation of a single aminoacid may have important consequences for binding affinity andother channel functions.

Recently, Opatowsky et al. [24] reported two stableinteracting domains within the β2 subunit; domain II (includingthe BID) is responsible for binding while domain I (N-terminalconserved domain) enhances binding. The in vivo interactionbetween α1C and β2 in the cardiovascular system also shouldbe analyzed because both types of subunit are reportedlyexpressed in heart [24,25]. If domain I cannot enhance bindingbetween the AID and domain II (which contains the BID),binding affinity might be reduced substantially. To date, onesplice variant that contains a portion of domain I (but notdomain II) has been described [22,25]. Additional studies arerequired to determine whether other splice variants exist and toanalyze the expression patterns of the various isoforms.

Only one neuronal isoform of the murine α1C gene has beenreported to date [6]. Our study revealed a novel cardiac and asmooth muscle isoform of α1C. At present, the mouse is themost important animal model in biological research because ofthe availability of transgenic mouse models. Therefore, cloningof the cardiac and smooth muscle α1C subunits may have agreat impact on studies of calcium channel physiology andcardiovascular research, and might also facilitate the develop-ment of therapies for cardiovascular disease that are based oncalcium channel antagonists. In humans, transcript scanning (avariant of PCR analysis) has revealed splice variants of α1C[26,27]. Our murine isoform, α1C-d, corresponds to the humansmooth muscle isoform that lacks exon 9*. The splice variant

isoforms that contain exon 9* are expressed selectively withinthe smooth muscle walls of arteries. In light of the −9 mVhyperpolarizing shift in voltage-dependent activation and the−11 mV shift in the current–voltage relationship of channelsthat contain α1C-d, alternative splicing appears to play animportant role in channel physiology, even though thesensitivity of the channels to blockade by nifedipine wasunaltered [26,27].

In conclusion, we identified a novel cardiac isoform of thevoltage-dependent calcium channel, α1C-a, and discovered anovel smooth muscle splice variant (α1C-d). The cardiacisoform exhibited preferential binding to the β2 subunit, whichmight promote the formation of long-lasting cardiac calciumchannels.

Acknowledgments

We thank Mrs. Toshihiro Mitsumori, Toshifumi Asano fortechnical assistance. This research was sponsored partly bygrants-in-aid from the Ministry of Education, Culture, Sports,Science and Technology of Japan.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.yjmcc.2006.05.002.

References

[1] Catterall WA. Structure and regulation of voltage-gated Ca2+ channels.Ann Rev Cell Dev Biol 2000;16:521–55.

[2] Mikami A, Imoto K, Tanabe T, Niidome T, Mori Y, Takeshima H, et al.Primary structure and functional expression of the cardiac dihydropyr-idine-sensitive calcium channel. Nature 1989;340:230–3.

[3] Bean BP. Pharmacology of calcium channels in cardiac muscle, vascularmuscle, and neurons. Am J Hypertens 1991;4:406S–11S.

[4] Welling A, Kwan YW, Bosse E, Flockerzi V, Hofmann F, Kass RS.Subunit-dependent modulation of recombinant L-type calcium channels.Molecular basis for dihydropyridine tissue selectivity. Circ Res 1993;73:974–80.

[5] Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, et al.Cav1.2 calcium channel dysfunction causes a multisystem disorderincluding arrhythmia and autism. Cell 2004;119:19–31.

[6] Ma W-J, Holz RW, Uhler MD. Expression of a cDNA for a neuronalcalcium channel α1 subunit enhances secretion from adrenal chromaffincells. J Biol Chem 1992;267:22728–32.

[7] De Waard M, Pragnell M, Campbell KP. Ca2+ channel regulation by aconserved beta subunit domain. Neuron 1994;13:495–503.

[8] Walker D, Bichet D, Geib S, Mori E, Cornet V, Snutch TP, et al. A newsubtype-specific interaction in α1A subunit controls P/Q-type Ca2+

chanõnel activation. J Biol Chem 1999;274:12383–90.[9] Baulard AR, Gurcha SS, Engohang-Ndong J, Gouffi K, Locht C, Besra

GS, et al. In vivo interaction between the polyprenol phosphate mannosesynthase Ppm1 and the integral membrane protein Ppm2 from Mycobac-terium smegmatis revealed by a bacterial two-hybrid system. J Biol Chem2003;278:2242–8.

[10] Murakami M, Ohba T, Xu F, Shida S, Satoh E, Ono K, et al. Genomicorganization and functional analysis of murine PKD2L1. J Biol Chem2005;280:5626–35.

[11] Flanagan JG, Leder P. The kit ligand: A cell surface molecule altered insteel mutant fibroblasts. Cell 1990;63:185–94.

[12] Soldatov NM. Genomic structure of human L-type Ca2+ channel.Genomics 1994;22:77–87.



[13] Biel M, Ruth P, Bosse E, Hullin R, Stuhmer W, Flockerzi V, et al. Primarystructure and functional expression of a high-voltage-activated calciumchannel from rabbit lung. FEBS Lett 1990;269:409–12.

[14] Biel M, Hullin R, Freundner S, Singer D, Dascal N, Flockerzi V, et al.Tissue-specific expression of high-voltage-activated dihydropyridine-sensitive L-type calcium channels. Eur J Biochem 1991;200:81–8.

[15] Pragnell M, De Waard M, Mori Y, Tanabe T, Snutch TP, Campbell KP.Calcium channel β-subunit binds to a conserved motif in the I–IIcytoplasmic linker of the α1-subunit. Nature 1994;368:67–70.

[16] De Waard M, Witcher DR, Pragnell M, Liu H, Campbell KP. Properties ofthe α1-β anchoring site in voltage-dependent Ca2+ channels. J Biol Chem1995;270:12056–64.

[17] Shistik E, Ivanina T, Blumenstein Y, Dascal N. Crucial role of N terminusin function of cardiac L-type Ca2+ channel and its modulation by proteinkinase C. J Biol Chem 1998;273:17901–9.

[18] Humme A, Delzeith O, Gomez SR, Moreno RL, Mark MD, Herlitze S.Competitive and synergistic interactions of G protein β2 and Ca2+ channelβ1b subunits with Ca(v)2.1 channels, revealed by mammalian two-hybridand fluorescence resonance energy transfer measurements. J Biol Chem2003;278:49386–400.

[19] Marquart A, Flockerzi V. α1-β interaction in voltage-gated cardiac L-typecalcium channels. FEBS Lett 1997;407:137–40.

[20] DeWaardM, Scott VES, PragnellM, Campbell KP. Identification of criticalamino acids involved in α1-β interaction in voltage-dependent Ca2+

channels. FEBS Lett 1996;380:272–6.

[21] Murakami M, Aoyama M, Suzuki T, Sasano H, Nakayama S, Iijima T.Genetic characterization of a new splice variant of the β2 subunit of thevoltage-dependent calcium channel. Mol Cell Biochem 2003;254:217–25.

[22] Murakami M, Yamamura H, Suzuki T, Kang MG, Ohya S, Murakami A,et al. Modified cardiovascular L-type channels in mice lacking thevoltage-dependent Ca2+ channel β3 subunit. J Biol Chem 2003;278:43261–7.

[23] Berrou L, Klein H, Bernatchez G, Parent LA. A specific tryptophan in theI–II linker is a key determinant of β-subunit binding and modulation inCaV2.3 calcium channels. Biophys J 2002;83:1429–42.

[24] Opatowsky Y, Chomsky-Hecht O, Kang M, Campbell KP, Hirsch JA. Thevoltage-dependent calcium channel β subunit contains two stable inter-acting domains. J Biol Chem 2003;278:52323–32.

[25] Harry JB, Kobrinsky E, Abernethy DR, Soldatov NM. New short splicevariants of the human cardiac Cavβ2 subunit: redefining the major func-tional motifs implemented in modulation of the Cav1.2 channel. J BiolChem 2004;279:46367–72.

[26] Tang ZZ, Liang MC, Lu S, Yu D, Yu CY, Yue DT, et al. Transcriptscanning reveals novel and extensive splice variations in human l-typevoltage-gated calcium channel, Cav1.2 alpha1 subunit. J Biol Chem 2004;279:44335–43.

[27] Liao P, Yu D, Lu S, Tang Z, Liang MC, Zeng S, et al. Smooth muscle-selective alternatively spliced exon generates functional variation inCav1.2 calcium channels. J Biol Chem 2004;279:50329–35.

identification of a cardiac isoform of the murine calcium channel α1c (cav1.2-a) subunit and its...

Documents