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Molecular and Cellular Biochemistry 254: 217–225, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Genetic characterization of a new splice variant ofthe βββββ2 subunit of the voltage-dependent calciumchannel

Manabu Murakami,1 Masahiro Aoyama,2 Takashi Suzuki,3

Hironobu Sasano,3 Shinnsuke Nakayama2 and Toshihiko Iijima1Department of Pharmacology, Akita University School of Medicine, Akita; 2Department of Cell Physiology, NagoyaUniversity Graduate School of Medicine, Shouwa, Nagoya; 3Department of Pathology, Tohoku University School ofMedicine, Aobaku, Sendai, Japan

Received 11 December 2002; accepted 14 April 2003

Abstract

This study reports a novel splice variant form of the voltage-dependent calcium channel β2 subunit (β

2g). This variant is com-

posed of the conserved amino-terminal sequences of the β2a

subunit, but lacks the β-subunit interaction domain (BID), whichis thought essential for interactions with the α

1 subunit. Gene structure analysis revealed that this gene was composed of 13

translated exons spread over 107 kb of the genome. The gene structure of the β2 subunit was similar in exon-intron organiza-

tion to the murine β3 and human β

4 subunits. Electrophysiological evaluation revealed that β

2a and β

2g affected channel prop-

erties in different ways. The β2a

subunit increased the peak amplitude, but failed to increase channel inactivation, while β2g

hadno significant effects on either the peak current amplitude or channel inactivation. Other β subunits, such as β

3 and β

4, signifi-

cantly increased the peak current and accelerated current inactivation. (Mol Cell Biochem 254: 217–225, 2003)

Keywords: voltage-dependent calcium channel, β subunit, gene structure, calcium current, subunit interaction domain

Introduction

Voltage-dependent calcium channels (VDCCs) are the mainpathways for calcium entry into cells, and play pivotal rolesin the control of calcium-linked cellular functions, such ascardiac muscle contractions. They are electrophysiologicallyand pharmacologically classified into five groups (L, N, T,R, and P/Q), and 10 genes for pore-forming α

1 subunits have

been reported [1]. L-type calcium channels are cruciallyimportant for excitation-contraction coupling, which acts asthe main pathway of calcium entry into the heart and smoothmuscle tissues, and are the targets of calcium channel blockers,such as DHPs, which are widely used in the treatment ofhypertension and angina pectoris. VDCCs have accessorycomponents, which include the α

2/δ, β and γ subunits [1]. The

β subunits are cytoplasmic proteins that increase current am-

plitude, accelerate the activation and inactivation of the cal-cium current, and increase channel populations [2–5]. Todate, four types of β subunit (β

1, β

2, β

3, β

4) have been reported.

Given its high-level expression in the heart, β2 is believed to

be the β subunit in cardiac calcium channels that couples withCa

v1.2b and composes L-type calcium channels [5–7]. The

high-affinity interaction between α1 and β occurs with a stoi-

chiometry of 1:1 and is dependent on the AID (α-subunitinteraction domain) and BID (β-subunit interaction domain)[8]. However, recent studies indicate the existence of otherinteraction sites [9], and reveal the functional importance ofβ

2a-specific palmitoylation of cysteines in the amino termi-

nal of the β2a

isoform for targeting of the channels to theplasma membrane [10–12]. Although a number of splice vari-ants of the β

2 subunit have been reported [5, 13, 14], most of

these studies concentrated on variant forms that had the BID.

Address for offprints: M. Murakami, Department of Pharmacology, Akita University School of Medicine, 1-1-1 Hondou, Akita 010-8543, Japan(E-mail: manabumurakami@excite.co.jp)

218

We report a novel splice variant form of the β2-subunit

gene, called β2g

. This variant isoform lacks the BID, but con-tains part of the highly conserved amino-terminal region. Weanalyzed the functionality of the new splice variant form ina cell line that expressed stably the cloned smooth muscle α

1

subunit (Cav1.2b), and compared its functions with those of

other β subunits.

Materials and methods

RNA isolation and RT-PCR (reverse transcriptase-polymerase chain reaction) analysis

Total RNA was isolated from the heart of a C57/BL6 mouseusing the RNeasy Kit (Qiagen Inc. Valencia, CA, USA). Thereverse transcription reaction was performed in a solution of10 pmol oligo-dT primer, 1 µg RNA, 1 × first strand cDNAbuffer (Life Technologies, Rockville, MD, USA), 10 mMdithiothreitol, 0.4 mM dNTPs, 40 U RNasin, and 200 U Su-perscript II in a volume of 25 µl, at 42°C for 45 min.

For the RT-PCR reaction, 2.0 µl of template were used. β2

subunit-specific sequences were amplified using primers β2-

1 (5′-ATGAAGGCCACCTGGATCAGGCTT-3′) and β2-2

(5′-TCATTGGCGGATGTATACATCCCT-3′), which corre-spond to the murine β

2 subunit sequences M1KATWIRL8 and

R605DVYIRQ611 and include the stop codon.

Tissue preparation and histology

Tissues were removed from wild-type mice under ether an-esthesia. The tissues were fixed immediately in 4% parafor-maldehyde in 0.1 M sodium phosphate buffer (pH 7.2) andincubated overnight at 4°C. The tissues were subsequentlyembedded in paraffin wax. Histological examinations wereperformed on hematoxylin-eosin-stained slides.

In situ hybridization

Probes that were specific for the β2a

-BID (β subunit interac-tion domain) and β

2g subunits were digoxigenin-UTP-labeled,

and transcripts were synthesized using T3 or T7 RNA polymer-ase from the murine β

2a-BID-specific sequence or the entire

β2g

clone. The murine β2a

-BID-specific sequence was ampli-fied by PCR with primers FV1 (5′-TWYWVNAAYGAY-TGGTGGATHGG-3′) and FV2 (5′-AANAANGCYTTYTTCATCATRTC-3′), which correspond to the F106NNDW-WIG113 and D250MMQKALF257sequences of the murine β

2a

isoform, respectively. The amplified fragment was subclonedinto the EcoRV site of the pBluescript vector (Stratagene, LaJolla, CA, USA).

In situ hybridization was performed using a manual capil-lary action system (MicroProbe Staining System; Fisher Sci-entific, Pittsburgh, PA, USA) with certain modifications.Tissue sections were rapidly dewaxed, cleared with alcohol,rehydrated in a Tris-based buffer (pH 7.4), and digestedwith pepsin for 3 min at 105°C. Each probe was added toformamide-free diluent, and the slides were heated to 105°Cfor 3 min, and allowed to hybridize at 45°C for 45–120 min.Hybridization times were modified empirically to detect strongsignals. The sections were washed 3 times with 2 × SSC(standard saline citrate) at 45°C and incubated with alkalinephosphatase-conjugated streptavidin. After two washes atroom temperature in AP chromogen buffer (pH 9.5), the hy-bridization products were visualized using fast-red salt. Theslides were counterstained with hematoxylin, air-dried, anda coverslip was placed on them for microscopic examination.

Electrophysiological analysis

Cell preparation

Chinese hamster ovary (CHO) cells that stably expressed thecloned smooth muscle α

1-subunits (Ca

v1.2b) (CHOCa9) [15,

16] were cultured in Dulbecco’s Modified Eagle’s Medium(DMEM), which was supplemented with 10% dialyzed fetalbovine serum, non-essential amino acids, streptomycin (30 µg/ml) and penicillin (30 units/ml). Cell passaging was carriedout when the cultures reached 80–90% confluence. For elec-trical recordings, CHOCa9 cells at 30–50% confluence wereplated onto 22-mm coverslips.

In some CHOCa9 cells, an expression vector that carriedthe β

2a, β

2g, β

3, or β

4 subunits [17, 18] was transfected along

with a GFP (green fluorescent protein) expression vectorusing lipofection (Superfect Transfection Reagent; Qiagen,Tokyo, Japan). Whole-cell patch clamp recordings were car-ried out 48–72 h after transfection. Expression of the β-subunits was quantified by the emission of green light usingan IX70 fluorescent microscope (Olympus, Tokyo, Japan).

Electrical recordings

A standard patch clamp technique was used to record mac-roscopic Ca2+ channel currents, as described previously [19].The patch clamp amplifier (Axopatch 200A; Axon Instru-ments, Foster City, CA, USA) was operated through an AD/DA converter (TL-1; Axon Instruments, Foster City, CA,USA). The measurements were carried out at room tempera-ture (22–26°C). Ba2+ (10 mM) was used as the charge carrier,and a cut-off frequency of 5 kHz was applied. The samplinginterval was usually set at 0.5 msec. The resistance of the pi-pette was approximately 5 MΩ, when a Cs+-rich pipetting

219

solution was used. The reference potential was set at zerocurrent potential before establishing the seal. Unless other-wise stated, the voltage of the cell membrane was clampedat –60 mV (holding potential). Capacitive transients werecompensated electrically. Normally, the CHOCa9 cells testedhad a membrane capacitance of 10–25 pF, and the series re-sistance was < 15 MΩ after applying series resistance com-pensation of 20–50%. Therefore, the time to clamped voltagewas < 0.3 msec.

Pooled data are shown as the mean ± S.E. of the mean. Sta-tistical significance was determined using Student’s unpairedt-test.

Results

Identification of transcripts of the murine cardiac β2

subunit by RT-PCR

Murine heart total RNA was reverse transcribed and ampli-fied by PCR using the specific primers β

2-1 and β

2-2. No ar-

tificial PCR products were amplified in the negative controlreaction, which contained the mixture used for PCR ampli-fication without the reverse transcribed product. Two prod-ucts, which were 492 and 1836 bp, were detected (Fig. 1).These fragments were excised from the gel, subcloned intoPCR 2.1-vector (Invitrogen, Carlsbad, CA, USA), and se-quenced. The 1836-bp product was found to be identical tothe murine β

2a subunit, as described in a previous report [20].

The 492-bp fragment was identified as a short form of the β2

gene, and was named β2g

. There was no evidence in the β2g

isoform of either a putative cAMP-dependent protein kinaseor protein kinase C phosphorylation sites.

Comparison of the amino acid sequences of four of the βsubunits

Figure 2A shows the alignment of the deduced amino acidsequences of β

2a and β

2g, which we discovered in this study,

and all known murine β subunit isoforms. The numbering atthe right in Fig. 2A indicates the corresponding amino acids.The alignment includes the sequences of the β

1 (accession

number NM-031173), β2a

[20], β3 [17], and β

4 [18] subunits.

There were two structurally conserved domains (boxed inFig. 2A), as reported previously [8]. The four β-subunit genesshared 64.2–64.8% identity for the N-terminal domain (Do-main I), 81.0–81.4% identity for the C-terminal domain (Do-main II), and 90.2% identity for the BID in the secondaryconserved domain (underlined in Fig. 2A). Taken together,the amino acid sequences of the murine β subunits havehighly conserved sequences in Domains I and II, but differat their amino termini, carboxyl termini, and at sequences

between the two conserved domains, as has been shown forother species [14]. The novel β

2g isoform has part of Domain

I, but lacks Domain II. The alignment was used to constructa phylogenetic tree (Fig. 2B).

Expression analysis of the β2 subunit by in situ

hybridization

The tissue-specific expression profiles of the β2a

-BID and theβ

2g subunit were examined by in situ hybridization analysis

of sections from the mouse aorta and heart (Fig. 3). Strongsignals for β

2a-BID and weaker signals for β

2g were detected

in both aortic smooth muscle cells (Fig. 3i) and heart ven-tricular myocytes (Fig. 3ii). Both isoforms were widely ex-pressed, although the hybridization signals were weak; thenovel β

2g isoform appears to be a minor transcript of the β2

subunit. In control experiments, hybridization was carried outon adjacent sections using the corresponding sense probes.All signals were equally distributed in both aortic smoothmuscle cells and heart ventricular myocytes.

Structure of the murine β2-subunit gene

The structure of the murine β2-subunit gene was character-

ized using the program BLAST in the NCBI database (http://www.ncbi.nlm.nih.gov/). One clone, which was derived frommouse chromosome 2A2 (accession number NW-000173),was found to be identical to the DNA sequence of the murineβ

2a subunit. Detailed sequence analysis of the exonic and most

of the intronic regions in the identified clone indicated thatthe coding region of the gene extended over ca. 108 kb, which

Fig. 1. Detection of β2g

- and β2a

-specific transcripts by RT-PCR. Identifi-cation of the 489-bp β

2g- and 1833-bp β

2a-specific transcripts in murine heart

by RT-PCR using primers β2-1 and β

2-2.

220

Fig. 2. Comparisons of the amino acid sequences of the mouse β subunits. (A) Comparison of the amino acid sequences and structural alignment of the β2g

isoform with other murine β subunits. Alignment was performed using the program ClustalX ver. 1.8. The amino acid positions are numbered in the rightmargin. Regions of highly conserved domains (Domains I and II) are boxed. The amino acids in the β-interaction domain (BID) are underlined. Asterisksindicate alignment of all four identical residues. The dashes represent gaps in the sequence. (B) Phylogenetic relationships between the murine calcium channelβ subunits.

221

is significantly longer than the coding region of the β3 subunit

(ca. 12 kb) [17]. The longest intron was the second one, whichlay between the second and third exons, and was > 75 kb inlength (Fig. 4A). The β

2-subunit gene contained 13 translated

exons, and existed as a single-copy locus in the haploid mousegenome. The sequences of all the exons and adjacent intronsare shown in Figure 4B.

Exon usage in the β2-subunit gene

Previous reports revealed that the β-subunit transcripts werespliced, and that splicing occurred preferentially at the cen-tral regions of the nucleotide sequence, between the twodomains [6, 13]. In order to analyze the splicing pattern ofthe murine β

2 subunit gene, we examined exon usage in the

β2g

variant (Fig. 5). The new splice variant contained exons1 and 2, and parts of exons 3 and 13, but lacked the interven-ing exons. Skipping exons 6, 7, and 8 results in the deletionof the BID domain in the β

2g variant. The exon-intron accep-

tor sequences are shown in Fig. 5b. There was no consensusGT-AG sequence at the start of the intron, while the spliceacceptor site was in good agreement with the AG sequence.

Electrophysiological recording of a transiently transfectedCa

v1.2b-expressing CHO cell line

Figure 6A shows the effects of the β-subunits on the whole-cell Ca2+ channel current. Rectangular pulses of –20, 0, and

+20 mV (200-msec duration) were applied. The top panelshows the current traces obtained from CHO cells that ex-pressed Ca

v1.2b alone (CHOCa9 cells), while the middle and

bottom panels show the current traces from CHOCa9 cellsthat co-expressed the β

2a or β

2g subunits, respectively. The

peak current density at 0 mV (PCD0mV

) was 14.6 ± 2.9 pA/pF in the CHOCa9 cells (n = 14). Co-expression of the β

2a

subunit significantly increased the peak amplitude to 31.8 ±9.0 pA/pF (n = 27). Co-expression of the β

2g subunit increased

the peak amplitude to 20.8 ± 3.1 pA/pF (n = 17), althoughthis increase was not statistically significant (p > 0.05). In ad-dition, β

2a and β

2g were co-expressed to examine whether β

2g

has an additional effect, such as a dominant-negative effect;β

2g had no significant effect on the increased current density

induced by β2a

. Since no prominent effect of β2 subunits was

found under our experimental conditions, we also examinedthe effects of the β

3 and β

4 subunits, which are known to

have strong effects on channels [5, 6, 21]. The β3 and β

4

subunits significantly increased the peak amplitudes in theCHOCa9 cells to 45.8 ± 12.7 (n = 17) and 31.9 ± 5.9 (n =14), respectively (Fig. 6B). There were no significant dif-ferences between the effects of the β

2a and β

2g subunits on

the inactivation percentages at 200 msec (Fig. 6C). In con-trast, the β

3 and β

4 subunits significantly accelerated the

inactivation rate.

Discussion

We discovered a novel splice variant form of the β2 gene,

called β2g

, which lacks the BID. In situ hybridization analy-ses showed strong, widely distributed expression of the BID-containing β

2 subunit form, and weak but widely distributed

expression of the β2g

isoform. Amino acid comparisons of allthe known murine calcium channel β subunits revealed twohighly conserved domains, as reported previously for otherspecies. We also clarified the structure of the β

2 gene. Our

electrophysiological data suggest that the novel β2 splice vari-

ant does not significantly affect channel properties.In this study, we tried to clarify the significance of the novel

β2 isoform (β

2g), but could not detect significant changes in

the peak currents, suggesting that this novel β2 isoform has

no special physiological function. There was no significantdifference in the peak amplitudes with β

2a and β

2g in the co-

expression experiment, suggesting that β2g

expression tendsto increase the current amplitude. This might occur becausethe effect of the new β

2 variant was slight and below the de-

tectable limit. Furthermore, we examined the co-expressionof β

2a and β

2g in CHO cells, to analyze if the novel isoform

has a dominant negative effect. The co-expression of β2a

andβ

2g had an intermediate effect on the current density between

the single expression of β2a

and β2g

, suggesting that β2g

prob-ably has no such effect. Although we chose stably Ca

v1.2b

Fig. 3. Results of in situ hybridization of the mRNA from β2-BID (A) and

β2g

(B) in the murine aortic smooth muscle layer (i, magnification ×200) andmurine heart ventricular myocytes (ii, magnification ×200). Hematoxylin wasused as the nuclear stain. The scale bars are 10 mm.

222

Fig. 4. Physical map of the murine calcium channel β2 subunit gene (A) and nucleotide and derived amino acid sequences of the translated exons (B). The

intron donor and acceptor sites are shown in lower case. The translated amino acid sequence is given above the nucleotide sequence of the coding exons insingle-letter code, and both sequences are numbered on the right. The numbering of the nucleotide sequence includes only the protein-coding nucleotides.The sequences of all exons and adjacent sequences were derived from those of mouse chromosome 2A2 (accession number NW-000173). Domains I and IIare underlined. The amino acids of the BID are boxed.

223

expressing CHO cells, which should have the same mRNAlevels, the differences in the currents between cells in whole-cell patch clamp recordings are relatively large, and this maymask the function of β

2g. Other methods of channel characteri-

zation, such as mRNA injection of β2a

into Xenopus oocytes,have shown that β

2a accelerates channel inactivation of

Cav1.2b [6], a phenomenon that was not seen in this study.

Therefore, it might be more advantageous to choose mRNAinjection into Xenopus oocytes as a functional assay for ac-cessory subunits, such as β, although the existence of endog-enous α1 and β subunits has been described and CHO cellsare apparently more suitable for physiological analysis, sincethey originated from a mammal.

To date, six different isoforms of the β2 gene have been re-

ported [14], three of which probably function as authentic β2-

subunit isoforms. These three isoforms have been isolatedfrom the rabbit heart and rat brain [6, 14]. They have differ-ent amino acid sequences at their amino terminals, whichsuggest the use of alternative exons, and they contain thehighly conserved Domains I and II. Of these three splicevariants, it is interesting that only the rat β

2a subunit has pal-

Fig. 5. (A) Distribution of the exons in the domain structure of the β2

subunit. The bar diagram represents the amino acid sequence of β2. The lo-

cations and boundaries of the exons are depicted by vertical lines. The barsbelow the bar diagram indicate highly conserved domains in all reported βsubunits (Domains I and II). The hatched bar indicates the BID. (B) Exon-intron boundary sequences of β

2g variant. There was no consensus GT-AG

terminal at the beginning of the intron sequence of the β2g

variant. The introndonor and acceptor sites are shown in lower case. The translated amino acidsequence is given under the nucleotide sequence of the coding exons insingle-letter code. Amino acid sequences in lower case are translated onlyin β

2a variant.

Fig. 6. (A) Representative current traces obtained from CHO cells thatexpress Ca

v1.2b alone (top), or β

2a- (middle) or β

2g- (lower) transfected cells.

The currents were evoked by depolarizing pulses to –20, 0, and +20 mVfrom a holding potential of –60 mV (200 msec duration). The dotted linesrepresent the zero current level. Leak currents were subtracted using a P/4protocol. The data were filtered at 200 Hz and digitized at 500 Hz. (B) Effectof different β subunits on the peak currents in CHOCa9 cells. Each columnand vertical bar represents the mean and S.E.M. from different CHOCa9cells, expressing Cav1.2 alone (n = 14), and co-expressing β

2a (n = 27), β

2g

(n = 17), β3 (n = 17), β

4 (n = 14), and β

2a and β

2g (n = 11) subunits. The

transfected β subunits are indicated at the bottom. *p < 0.05 vs. Cav1.2.(C) Effect of different β subunits on inactivation at 200 msec in CHOCa9cells. The ratio of inactivation at 200 msec (voltage at 0 mV) was calcu-lated by setting the peak current density at 0 mV as 1.0. Each column andvertical bar represents the mean and SEM from different CHOCa9 cells,expressing Cav1.2 alone (n = 14), and co-expressing β

2a (n = 27), β

2g (n =

17), β3 (n = 17), β

4 (n = 14), and β

2a and β

2g (n = 11) subunits. Data are ex-

pressed as means ± S.E.M. The transfected β subunits are indicated at thebottom. *p < 0.05 vs. Cav1.2.

224

mitoylation-sensitive cysteines, which take part in the traf-ficking of calcium channels [10]. Conversely, there are threenon-functioning alternative splice variant forms [14]. Theirsequences contain frame-shifts, which result in truncation,and they lack the BID. The β

2g isoform is interesting in that

it has no BID and no palmitoylation-sensitive cysteines at theamino terminus, but it has a part of Domain I, which has atendency to increase calcium currents. It would be quite in-teresting to discover the functions of these β subunits that lackBID, as they might affect channel functions without bindingα1 subunits. Even if the β

2g isoform lacks functionality as a

channel component, it might affect the regulation of chan-nel gene expression. Since the existing analytical approachesfor auxiliary channel subunits are limited and the assays showextreme variation, new systems of analysis will have to bedeveloped to clarify the fine effects of various splicingisoforms and to improve channel characterization.

Since a number of splice variants of the β2 subunit gene

have been described in other species [13, 14], it seemed likelythat β

2-subunit isoforms are important factors that determine

the kinetics of L-type calcium channels in the heart. Alter-native splicing and expression in the heart of the β

3 subunit

is already known. Mermelstein et al. reported that β-subunitisoforms defined the biophysical characters of the α

1A subunit,

namely the P- and Q-type channels [22]. In that study, differ-ences in the inactivation kinetics of neostriatal neurons wereattributed to the β subunits, and β

2a was responsible for the

slow (P-type-like) inactivation kinetics of some Q-type chan-nels. Different subcellular distributions of the β

2a (soma) and

β3 (dendrites) subunits in cerebellar Purkinje neurons have

also been described [23]. The differential expression of thetwo β subunits and their size heterogeneity suggest the ex-istence of multiple splicing variants. Channel kinetics andsubunit populations in the heart are probably defined by twofactors: accessory subunits and splicing events.

The chaperoning effect of the β subunits in transport to theplasma membrane is well known [24, 25]. This effect is thoughtto be dependent mainly on the interaction between the AIDand BID of the α

1 and β subunits, and causes an increase in

the channel population at the cell membrane. In this study,we found that the β

2a subunit had a significant effect only on

the peak amplitude, and did not affect channel inactivation,whereas the β

3 and β

4 subunits had a significant impact on

the peak amplitudes and increased the inactivation rates.These different effects on channel function by different βsubunits might be advantageous for some physiological proc-esses, such as heart muscle contraction. Heart muscles needmore prolonged calcium-influx for blood pumping than otherorgans, such as the neuronal systems, where local, subcellu-lar, high-level concentrations of calcium and rapid regulationof synaptic neurotransmission are essential.

This study is the first to examine the amino acid sequencesof all known murine β subunits. The β-subunit amino acidsequences of mice were highly conserved, as in other spe-cies, with two highly conserved domains and very highlyconserved BID sequences [8, 14, 21]. The high-level conser-vation of the calcium channel β subunits suggests that theyhave important physiological roles, and emphasizes the valueof mutant mouse models in the analysis of channel functions.Furthermore, the β

2 subunit, like the β

3 subunit and other ba-

sic channel subunits, is highly conserved evolutionarily.This study reports the genetic organization of the murine

calcium channel β2 subunit. The gene contains 13 translated

exons that extend over 100 kb. The sizes and distribution ofthe exons and introns were evaluated using the mouse ge-nome database. Our study demonstrated that the genetic struc-ture of the β

2 subunit is essentially the same as that of the β

3

subunit, which also has 13 exons [17]. It is noteworthy thatthe human β

4 gene has 13 exons and a similar gene structure

[26]. The conserved domains of the murine β2 gene were very

similar to those of the β3 gene. Domain I consisted of three

exons (2, 3, and 4) and Domain II consisted of 7 exons (6–12). Our analysis demonstrates that the divergent amino ter-mini and carboxyl termini are contained in exon 1 and exon13, respectively, as is the case for the β

3 gene. The most highly

conserved sequences were found in exons 7 and 8, in whichBID is encoded with very high levels (over 90%) of aminoacid identity among all the known β subunits. Since this BIDsegment [8] is highly conserved, not only at the amino acidsequence level, but also with the same exon-intron usagepattern (exons 7 and 8, as for the β

3 gene), the second con-

served domain (including BID) probably represents the coredomain, which has evolved from an ancestral β-subunit gene.Conversely, the very long intron sequence (> 75 kb) betweenexons 2 and 3 suggests that the β

2-subunit gene has some spe-

cialized features. Several amino-terminal splice variants ofthe β

2 subunit have been reported in other species [14]. There-

fore, it is possible that this long intron includes several exonsof the β

2 splice variants. However, this is probably not the

case, since exon 2 contains a part of the highly conservedDomain I sequence.

Furthermore, we expected to uncover the function of theother highly conserved domain (Domain I), whose sequenceis also partly included in the new β

2 variant. Domain I is

thought to influence inactivation [27]. However, we foundno significant effects on channel inactivation, even with theβ

2a form. Therefore, it was reasonable that we could not de-

tect a significant influence of the new splice variant on chan-nel inactivation under our experimental conditions.

In conclusion, we discovered a novel β2-subunit splice vari-

ant of the voltage-dependent calcium channels, and we char-acterized the gene for the murine calcium channel β

2 subunit.

225

Acknowledgements

We would like to thank Mr. Hirotaka Tanabe for his techni-cal assistance. This research was sponsored partly by grants-in-aid from the Ministry of Education, Science, and Culture,of Japan, and from the Japan Foundation for CardiovascularResearch.

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