Cloning of a calcium channel α1 subunit from the reef-building coral, Stylophora pistillata

Gene 227 (1999) 157–167

Cloning of a calcium channel a1 subunit from the reef-building coral, Stylophora pistillata Didier Zoccola a,1, Eric Tambutte´ a,2, Franc¸oise Se´ne´gas-Balas b, Jean-Franc¸ois Michiels c, Jean-Pierre Failla c, Jean Jaubert a, Denis Allemand a,* a Observatoire Oce´anologique Europe´en, Centre Scientifique de Monaco, Avenue Saint Martin, MC-98000 Monaco, Monaco b Laboratoire d’Histologie, Faculte´ de Me´decine, Avenue de Valombrose, F-06107 Nice, Cedex 2, France c Laboratoire d’Anatomo-Pathologie, Hoˆpital Pasteur, Avenue de la Voie Romaine, F-06000 Nice, France Received 26 December 1997; received in revised form 25 November 1998; accepted 5 December 1998

Abstract While the mechanisms of cellular Ca2+ entry associated with cell activation are well characterized, the pathway of continuous uptake of the large amount of Ca2+ needed in the biomineralization process remains largely unknown. Scleractinian corals are one of the major calcifying groups of organisms. Recent studies have suggested that a voltage-dependent Ca2+ channel is involved in the transepithelial transport of Ca2+ used for coral calcification. We report here the cloning and sequencing of a cDNA coding a coral a1 subunit Ca2+ channel. This channel is closely related to the L-type family found in vertebrates and invertebrates. Immunohistochemical analysis shows that this channel is present within the calicoblastic ectoderm, the site involved in calcium carbonate precipitation. These data and previous results provide molecular evidence that voltage-dependent Ca2+ channels are involved in calcification. Cnidarians are the most primitive organisms in which a Ca2+ channel has been cloned up to now; evolutionary perspectives on Ca2+ channel diversity are discussed. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Biomineralization; Cnidaria; Protein evolution; Transepithelial calcium transport

1. Introduction Ca2+ ion is not only a regulatory agent in physiological processes but also the primary cation used in biomineralized structures in plants, invertebrates and vertebrates (Simkiss and Wilbur, 1989). While small, rapid and transitory Ca2+ uptake through Ca2+ channels appears sufficient to trigger a wide variety of intracellular processes, the continuous transport of large amounts of Ca2+ is needed to build a skeleton. But although a large * Corresponding author. Tel: +377 9330 1211; Fax: +377 9350 5297; e-mail: allemand@ unice.fr 1 Present address: UMR 6549 CNRS, Faculte´ de Me´decine, Avenue de Valombrose, F-06107 Nice Cedex 2, France. 2 Present address: Service de l’environnement, 3 avenue de Fontvieille, MC-98000 Monaco, Principality of Monaco. Abbreviations: A Ec, aboral ectoderm; A En, aboral endoderm; bp, base pairs; Co, coelenteric cavity; DHP, dihydropyridine; O Ec, oral ectoderm; O En, oral endoderm; ORF, open reading frame; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; STPCACH, Stylophora pistillata calcium chanel; Z, zooxanthellae.

amount of information has been produced in the last few years on biomineralization processes, the mechanism of Ca2+ transport largely remains a biological enigma (Bawden, 1989; Lundgren and Linde, 1997). In both intracellular or extracellular biomineralization processes, the primary event is the entry of Ca2+ ions at the cell membrane level. On the basis of pharmacological evidence, the involvement of verapamil-sensitive Ca2+ channels has been suggested in a few calcifying marine invertebrates such as echinoids (reviewed in Dubois and Chen, 1989), crustaceans (Ahearn and Zhuang, 1996) and corals (Allemand and Grillo, 1992; Marshall, 1996; Tambutte´ et al., 1996). Dihydropyridines have been shown to suppress the uptake of Ca2+ ions during both osteogenesis (Duriez et al., 1993) and dentinogenesis (Lundgren and Linde, 1997) in vertebrates. However, only one calcium channel has been cloned up to now in a calcifying cell, the osteoblast (Meszaros et al., 1996) and this L-type calcium channel is more probably involved in calcium signalling rather than in the calcium transport process for calcification (Meszaros et al., 1996).

0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 60 2 - 7

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Scleractinian corals (Cnidarian, Anthozoa) are one of the major group of calcifying animals. The rate of calcification of a tropical coral reef is assumed to be around 10 kg CaCO /m2 year (Chave et al., 1975), 3 consequently coral reefs play a major role in the biogeochemical cycle of calcium carbonate. Aragonitic coral skeleton is produced externally by the calicoblastic (ectodermal ) epithelium (Johnston, 1980). Recent pharmacological studies performed in the scleractinian corals Galaxea fascicularis (Marshall, 1996) and Stylophora pistillata ( Tambutte´ et al., 1996) have demonstrated that calcification in these animals is inhibited by antagonists of L-type calcium channels, suggesting that a voltagedependent calcium channel is involved in the transepithelial transport of Ca2+ used for coral calcification. Despite the fact that knowledge of the molecular biology of voltage-dependent calcium channels in invertebrates is increasing rapidly, only few molecular mechanisms have been elucidated up to now (see Skeer et al., 1996 for review). Several types of voltage-gated Ca2+ channels (L, T, N, P/Q and R types) have been identified primarily in excitable mammalian cells by functional criteria. Cloning of the different subunits (a1, a2, b, c and d) composing these channels revealed the major role played by the a1 subunit in voltage dependence as well as in the specificity of the channel for calcium ions and sensitivity to various pharmacological agents (see Perez-Reyes and Schneider, 1994 for review). Two structural subfamilies of a1 subunits have emerged from molecular cloning of mammalian cDNAs: the L-type subfamily, which is pharmacologically modulated by dihydropyridines (DHP) (including a1C, a1D and a1S subunits), and the nonL-type subfamily, which is insensitive to DHP (including P/Q type a1A, N-type a1B and R-type a1E subunits). In the present study, we develop a molecular approach to characterize the Ca2+ channel involved in the calcification process in the scleractinian coral, Stylophora pistillata. The cloning and sequencing of the complementary DNA encoding a1 subunit of this Ca2+ channel are reported. A comparative analysis between this channel and the others cloned so far in various species is presented. An immunohistochemical analysis was used to localize this channel in coral tissue.

2. Materials and methods 2.1. Biological material Cloned microcolonies were propagated in the laboratory as previously described ( Tambutte´ et al., 1996) from small fragments of the scleractinian coral Stylophora pistillata. Briefly, terminal portions of branches (6–10 mm long) were cut from parent colonies and either placed on a nylon net (1 mm×1 mm mesh)

or fixed on a glass slide using a marine epoxy resin ( UW paste, DEVCON Ltd., Ireland ). Parent colonies and microcolonies were stored in an aquarium supplied with Mediterranean seawater (exchange rate 2%/h), heated to 26.0±0.1°C and illuminated with a constant irradiance of 175 mmol photons/m2 s. 2.2. RT–PCR Reverse transcription experiments were performed using oligo-(dT ) or specific primers (see below) with the RT–PCR kit according to the instructions of the supplier (Stratagene, La Jolla, CA, USA). PCR experiments were carried out as described in Table 1 using Taq DNA polymerase (Boehringer Manheim, Germany) on DNA thermal cycler 480 (Perkin Elmer, Foster City, CA, USA). Each PCR amplification product was cloned and five independent clones from two unrelated amplifications were sequenced at least, thus reducing sequence errors resulting from PCR. 2.3. Isolation and characterization of the cDNA An oligo-dT primed cDNA library was constructed in lambda ZAPA II (Stratagene) using 5 mg poly(A)+ RNA isolated from Stylophora pistillata colony by the Chomczynski method (Chomczynski and Sacchi, 1987), followed by one passage over oligo(dT )-cellulose columns and the Great Lengths@ cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA). It was screened with a cDNA probe obtained by degenerate oligonucleotide PCR after reverse transcription of coral mRNA. Primers ( FPA and RPA) (Horne et al., 1993) were synthesized based on amino sequences IGMQ( V/M )FG and VAVIMDNF localized in the highly conserved regions in the IV S5 and IV S6 domains of previously cloned a1 subunits. After three rounds of positive hybridization, in vivo excision of the recombinant pBluescript SK(−) phagemid from the lambda ZAPA II clones was performed. DNA sequencing was carried out on both strands by the dideoxy chain termination method using a T7 sequencing kit (Pharmacia, Uppsala, Sweden). Oligonucleotide sequences were chosen in 5∞ of the first clone and used, together with degenerate primers located in upstream potentially conserved regions, in PCR experiments ( Table 1). For the first round, a specific primer (RPB1) located at amino acid position 1170–1176 and a degenerate primer ( FPB) corresponding to the pore region (P loop) between the IIS5 and IIS6 segments were used. The second round was performed with a specific primer (RPB2) located at amino acid position 1102–1108 and the same degenerate primer. By cloning (pTAg vector, R and D Systems) and sequencing the amplified products, we obtained a 1378 bp fragment including 1317 bp of a new sequence located 5∞ of the first one obtained. The same strategy

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D. Zoccola et al. / Gene 227 (1999) 157–167 Table 1 Primers and conditions used in PCR analysis Name

Sequence

Conditions for assay

FPA RPA

5∞-ATHGGIATGCARRTITTYGG-3∞ 5∞-TAITCRAARTTRTCCATDATIAC-3∞

FPB RPB1

5∞-ACNGGNGARGAYTGGAA-3∞ 5∞-CGAATATCAGATATTCAAAGGC-3∞

RPB2

5∞-GACCATAAAGAAGGCAATGAT-3∞

FPC RPC1

5∞-GYATHACNHTNGARGGNTGGAC-3∞ 5∞-AACTGGAAGAGCATGGTAAC-3∞

RPC2

5∞-TCTCCAATGCCACCCCATGC-3∞

RPD1 RPD2

5∞-CTGTCGCCTTCAATGTCCTCA-3∞ 5∞-CGACTTGCTGCTTTTCTCTAA-3∞

RPD3

5∞-TGTCGTCTCGCCTTTTCTTTG-3∞

FPA/RPA, 3 mM MgCl 2 35 cycles of 1 min at 94°C, 2 min at 48°C, 1 min at 72°C FPB/RPB1, 3 mM MgCl 2 35 cycles of 1 min at 94°C, 2 min at 48°C, 1 min at 72°C FPB/RPB2, 1.5 mM MgCl 2 25 cycles of 1 min at 94°C, 2 min at 48°C, 1 min at 72°C FPC/RPC1, 3 mM MgCl 2 35 cycles of 1 min at 94°C, 1 min at 52°C, 2 min at 72°C FPC/RPC2, 1.5 mM MgCl 2 25 cycles of 1 min at 94°C, 1 min at 52°C, 2 min at 72°C Oligod( T )-anchor/RPD2 Standard conditions, according to the supplier’s instructions Anchor/RPD3 Standard conditions, according to the supplier’s instructions

Note: H=A, T or C; I=Inosine; R=A or G; Y=C or T; D=G, A or T; N=A, G, C or T.

was reproduced with specific primers RPC1 and RPC2, and a degenerate primer (FPC ) corresponding to the P loop between IS5 and IS6 segments, and an additional fragment of 1071 bp including 1031 bp of 5∞ sequences was again obtained. To obtain the NH -terminus coding 2 sequences, we used Rapid Amplification of cDNA Ends (RACE) experiments (5∞/3∞ RACE Kit; Boehringer Manheim). RPD1 primer located in the 5∞ cloned region (amino acid position 1279–1299) was used for the reverse transcription of polyadenylated mRNAs isolated from coral colonies. The single-strand cDNA obtained was tailed with a polyA track and PCR was performed using RPD2 oligonucleotide located at the amino acid position 1218–1238 and a primer containing an anchor sequence and a polyT track complementary to the A tail artificially added at the 5∞ extremity of the cDNA. A second nested PCR was performed with RPD3 primer located at the amino acid position 1174–1194 and the anchor primer. This technique produced a fragment of 1238 bp which was cloned in pTAg vector and sequenced as mentioned above. 2.4. Immunohistochemistry S. pistillata colonies cultured on glass slides were fixed in 4% paraformaldehyde in 0.6 M KCl (1200 mOsm/l ). They were then decalcified using Rapid Bone Decalcifying product ( Eurobio), dehydrated and embedded in paraffin. We used as primary antibody, the MA3-920 mouse monoclonal antibody raised against rabbit skeletal muscle DHP receptor a1 subunit (ABR,

Inc.). This antibody is able to inhibit the DHP-sensitive calcium current in mouse muscle cells (Morton et al., 1988). Cross-sections of 3 mm were sequentially incubated with primary mouse monoclonal anti-DHP receptor (24 h, 4°C, 1/20 in phosphate-buffered saline (PBS) pH 7.2, 0.05 M ) and a peroxidase conjugated anti-mouse antibody (2 h, room temperature, 1/50 in PBS pH 7.2, 0.05 M ). The site of antibody binding was visualized by incubation in diamino-benzidine (Sigma, St Louis, MO, USA). The slides were counterstained with Harris’ haematoxylin. Several controls were routinely performed: (a) PBS; or (b) monoclonal mouse anti-bromo deoxyuridine (DAKO); and (c) staining with only diaminobenzidine.

3. Results 3.1. cDNA cloning The first step of cloning consisted of the construction of a cDNA library corresponding to polyadenylated messengers from the coral Stylophora pistillata (see Materials and Methods). Three different types of probes were found. Databank comparisons (FASTA program) indicated that two of them corresponded to N-like (p=2.0 e−24 and 64% homology with channel M94173) and P-like (p=1.7 e−21 and 76% homology with channel M64373) types of calcium channels. A probe corresponding to a L-like-type channel was also obtained. This specific probe was then used to pick up a clone

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Fig. 1. Amino acid alignment of Stylophora pistillata a1 subunit Ca2+ channel, accession number U64465 (STPCACH ) with rabbit a1C subunit, accession number X15539 (RABa1C ). Dashed lines represent gaps introduced by the CLUSTAL program to optimize alignment. Identical amino acid residues are indicated by ‘*’ and amino acid conservative substitutions by ‘.’. The four domains containing six putative membrane spanning regions are marked with solid lines and labelled IS1 to IVS6. The pore region is labelled ‘P loop’. The putative N-glycosylation sites are shown as ‘‡’ and the putative cAMP-dependent protein kinase phosphorylation sites as ‘1’.

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from the specific coral cDNA library. This clone contained a 4146 bp insert with a potential open reading frame (ORF ) of 768 amino acids and 1842 bp of a noncoding region corresponding to the most 3∞ of the messenger. From this first clone, 5∞ oligonucleotide sequences were chosen and used, together with degenerate primers located in potentially conserved upstream regions, in PCR experiments ( Table 1). By cloning and sequencing the amplified products, we obtained a new sequence located upstream. The same strategy was repeated and an additional fragment was again obtained. The last fragment that we obtained by RACE experiments ( Frohman et al., 1988) was 1238 bp long and contained at the 5∞-end 110 bp of non-coding sequence with stop codons in three frames. From the sequences obtained by the first clone and the PCR products, computer analysis revealed, at 110 pb from the 5∞-end, a methionine in a Kozak’s context ( Kozak, 1984), followed by an ORF of 5673 bp which codes for a protein of 1891 amino acids and a calculated molecular mass of 213 kDa. Fig. 1 shows the amino acid sequence of the Stylophora pistillata Ca2+ channel (STPCACH ) as predicted from the 7517 bp cloned cDNAs. The structure of this a1 subunit is typical of a voltage-dependent calcium channel. It contains four repeated units of homology (amino acid residues 72–351, 469–704, 850–1115 and 1169–1411). Each repeat has five hydrophobic segments (S1, S2, S3, S5 and S6) and one positively charged segment (S4) with a conserved Arg or Lys at every third position. Identities and conservative substitutions between the rabbit a1C subunit and the S. pistillata Ca2+ channel are 52.5% and 86%, respectively. The four repeat domains show a higher identity (I: 69%, II: 67%, III: 64%, IV: 60%) than regions such as the cytoplasmic COOH-terminus region (49%). In the structural model given in Fig. 1, two of the twelve potential N-glycosylation sites (Asn 248 and 874) are located on the external surface. Amino acid analysis reveals several putative phosphorylation sites for cAMP-dependent kinase (four sites: Ser 412, Thr 460, Ser 1676 and Ser 1677), protein kinase C (17 sites), or tyrosine kinase (one site: Tyr 373). Futhermore, primary structure shows that the I–II cytoplasmic linker contains the conserved amino-acid sequence, sufficient for the binding of the calcium channel b-subunits (Pragnell et al., 1994). 3.2. a1 subunit encoded by STPCACH cDNA belongs to the L-type family In order to classify the Stylophora pistillata Ca2+ channel as an L-type or non-L-type, we compared it with previously cloned a1 subunits (Perez-Reyes and Schneider, 1994; Grabner et al., 1994; Wilson et al., 1994; Zheng et al., 1995; Smith et al., 1996; Jeziorski et al., 1998). By using clustalW program ( Thompson

et al., 1994), alignment of complete amino acid sequences of the proteins indicated that the best identity is obtained with Cyanea a1 subunit (56.6%) and rabbit a1C (52.5%), whereas less identity is obtained with Drosophila melanogaster a1 ( U55776) and rabbit a1A, a1B and a1E (40%; 41.9%, 42.2% and 42.8%, respectively). The PILEUP program was used to generate and plot a dendrogram ( Fig. 2) which shows the clustering relationships used to obtain the alignments. This dendrogram indicates a separate branching between the L-type and the non-L-type subfamily and shows that the coral calcium channel belongs to the L-type subfamily. However, the separate branching between the S. pistillata Ca2+ channel and all the L-type subfamily channels does not permit us to classify the coral channel among one of the three subtypes known in mammals. To confirm that the coral subunit belongs to the L-type subfamily, we compared the putative DHP binding site of the coral subunit with the rabbit ones. The IIIS6 and IVS6 domains are important regions of high affinity binding of DHP agonists and antagonists to L-type calcium channels (Grabner et al., 1996; Peterson et al., 1997). The sequence aligment of IIIS6 and IVS6 domains of STPCACH a1 subunit (Fig. 3) shows a higher identity with the rabbit L-type subunits than with the non-L-type subunits (77% vs 42% in the IIIS6 region). Recently, using site-directed mutagenesis studies, Peterson et al. (1997) have identified aminoacid residues within transmembrane segments IIIS6 and IVS6 whose mutation has significant effects on DHP binding affinity. In segment IIIS6, Tyr1099, Ile1100, Ile1103 are conserved in S. pistillata a1 subunit. However, a Val1108, instead of Met, is present. In segment IVS6, the crucial Tyr1397 and Ile1405, Asn1406 are conserved. However, a Ser1398, instead of Met, is present.

3.3. Localization of the coral a1 calcium channel The IIIS6 and IVS6 regions involved in DHP-binding determined in rabbit a1 subunit share 80% homology with our cloned a1 subunit (Fig. 3). A monoclonal mouse antibody, directed against these rabbit regions (Morton and Froehner, 1987) and able to detect mouse and human a1 subunits (Morton et al., 1988, 1994), was used in an immunocytochemical assay in order to locate the channel. If the protein encoded by our cloned cDNA is involved in the calcification process, we can presume that it should be at least located within the epithelial cell layer facing the skeleton, i.e. the calicoblastic ectoderm (Johnston, 1980). Anti DHP-receptor antibody staining shows that the L-type channel is located as expected within the calicoblastic ectoderm (Fig. 4B and D) and within the oral ectoderm ( Fig. 4B and C ). All control tests were negative ( Fig. 4A).

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Fig. 2. Dendrogram showing sequence relationships between the Stylophora pistillata a1 subunit calcium channel and other cloned a1 subunits. Homologies between full-length amino acid sequences were calculated using the GCG PILEUP program. Distances between STPCACH and other channel amino-acid sequences ( labelled in bold ) were calculated using a neighbour-joining algorithm as implemented in ClustalW (ignore gaps= off; multiple substitutions=off ). Accession number channel sequences found in the Genbank database (National Center for Biotechnology Information) are given in parentheses.

4. Discussion We report here cloning and sequencing of a cDNA coding for a coral L-type a1 subunit Ca2+ channel. The a1 subunit is an integral membrane protein which contains the receptor binding site for dihydropyridines and other Ca2+ entry blockers (benzothiazepines, phenylal-

kylamines) and is sufficient to direct the permeation of Ca2+ in a voltage-dependent manner (Catterall, 1988; Perez-Reyes et al., 1989). Our results provide the first molecular characterization of a calcium channel probably involved in biomineralization in a marine invertebrate. Together with the Ca2+ channel a1 subunit recently cloned in another Cnidarian, the jellyfish,

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Fig. 3. Sequence alignment of transmembrane segment IIIS6 (A) and IVS6 (B) from DHP-sensitive and DHP-insensitive rabbit a1 subunit Ca2+ channel and STPCACH. Shaded boxes refer to amino-acid residues conserved in the different types. Also indicated by asterisks, amino acids important for DHP-binding defined by Peterson et al. (1997).

Cyanea capillata (Jeziorski et al., 1998), the epithelial scleractinian coral Ca2+ channel a1 subunit is, evolutionary, the most distant of the sequences described to date. These results confirm radiotracer flux studies which have demonstrated that calcium deposition in the scleractinian coral, Stylophora pistillata, is dependent on a calcium channel sensitive to dihydropyridines, phenylalkylamines and benzothiazepines (Tambutte´ et al., 1996) resembling the L-type voltage-sensitive Ca2+ channel described in mammalian cells (Hosey and Lazdunski, 1988). The presence of voltage-dependent Ca2+ channels is widely documented in vertebrates (Perez-Reyes and Schneider, 1994) and has been demonstrated by pharmacological studies in numerous marine invertebrates

( Hagiwara et al., 1975; Adams and Gage, 1979; Bilbaut et al., 1988). The involvement in the calcification process of verapamil-sensitive Ca2+ channels is less documented and has only been suggested in echinoids (Dubois and Chen, 1989), crustaceans (Ahearn and Zhuang, 1996), anthozoans (Allemand and Grillo, 1992; Marshall, 1996; Tambutte´ et al., 1996), as well as in vertebrates for osteogenesis (Duriez et al., 1993) and dentinogenesis (Lundgren and Linde, 1997). In addition to our previous physiological results ( Tambutte´ et al., 1996), present data, demonstrating the presence of an L-type a1 subunit calcium channel and its immunolocalization within the calicoblastic epithelium responsible for skeletogenesis (Johnston, 1980), strongly argue in favour of the involvement of the cloned voltage-dependent calcium

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Fig. 4. Immunolocalization of DHP receptors in Stylophora pistillata. (A) Representative section showing both oral epithelium with ectoderm (O Ec) and endoderm (O En) containing zooxanthellae (Z ) and aboral epithelium with endoderm (A En) and ectoderm (A Ec), also called calicoblastic ectoderm. Oral and aboral epithelia are separated by a coelenteric cavity (Co). This section was incubated with mouse monoclonal anti-bromouridine as primary antiserum (control ) (scale bar: 60 mm) and shows no labelling. (B) Incubation of S. pistillata section with monoclonal antiDHP receptor antibody showing labelling of both O Ec cells and A Ec cells indicated by arrows (scale bar: 70 mm). (C ) Anti-DHP receptor antibody clearly stained the apical plasmic membrane of O Ec cells (scale bar: 240 mm). (D) Anti-DHP receptor antibody also labelled the A Ec cells (scale bar: 240 mm). This labelling method demonstrated the presence of calcium channel in O Ec cells and A Ec cells.

channel in transepithelial calcium transport for calcification in hermatypic corals. Such channels could be responsible for the passive entry of Ca2+ into calicoblastic cells through which the bulk, if not all, skeletal Ca2+ must pass. The additional localization of the Ca2+ channel in oral ectoderm agrees with our previous studies ( Tambutte´ et al., 1996) which showed that a tissue compartment different from the calicoblastic ectoderm and sensitive to DHP was present in S. pistillata. Our results indicate that this compartment is the oral ectoderm. Because they display a slow inactivation (half-time: 20–100 ms) and short mean open time (0.5–20 ms), L-type Ca2+ channels appear to be involved primarily in the regulation of intracellular Ca2+ homeostasis and

Ca2+ signalling in a wide range of tissue including nonexcitable cells ( Hosey and Lazdunski, 1988). During the calcification process, continuous transport of a large amount of calcium is necessary. Similar vectorial transport of calcium occurs in epithelial cells of vertebrate kidney distal convoluted tubule ( Yu et al., 1992; Matsunaga et al., 1994) or small intestine ( Van Os, 1987). Surprisingly, underlying molecular mechanisms of this transport are poorly understood and only partial sequences have been available up to now for calcium channels mediating transcellular transport in epithelial cells ( Yu et al., 1992; Yu, 1995). The epithelial coral Ca2+ channel a1 belongs to the same supergene family as mammalian and insect a1 subunits involved in Ca2+ signalling. The subunit

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described here shows the same four repeat structures, each containing six transmembrane segments, which is the characteristic pattern for voltage-dependent Ca2+ and Na+ channels (Catterall, 1988). Concerning the DHP-binding region, sequence aligment shows that the coral subunit is closer to the L-type family than the non-L-type ( Fig. 2). Only two amino-acid discrepancies were found within the residues defined by Peterson et al. (1997) as essential for DHP-binding. Despite these differences, it should be noted that the coral Ca2+ channel is sensitive to the DHP antagonist, nifedipine and insensitive to the DHP agonist, Bay K 8644 ( Tambutte´ et al., 1996). Analysis of the S. pistillata Ca2+ channel a1 subunit provides some evolutionary perspectives on Ca2+ channel diversity. Only few Ca2+ channels have been cloned in invertebrates: one within the phylum Nematoda (Caenorhabditis elegans, Wilson et al., 1994) and two within the phylum Arthropoda (housefly: Grabner et al., 1996 and Drosophila melanogaster: Zheng et al., 1995; Smith et al., 1996). Together with the recently cloned Ca2+ channel a1 subunit in the jellyfish Cyanea capillata (Jeziorski et al., 1998), the present coral a1 subunit is the more primitive invertebrate Ca2+ channel studied until now. The a1 subunit Ca2+ channel dendrogram (Fig. 2) is consistent with the accepted divergence of cnidarians early in metazoan evolution. However, to further substantiate this conclusion, more invertebrate genes need to be cloned. Based on 18S rRNA data it has been suggested that cnidarians were not monophyletic with higher metazoans ( Field et al., 1988). The high degree of sequence similarity between coral and vertebrate a1 subunit Ca2+ channel found in the present study together with the high similarity between cnidarian and human integrin b1 subunits (Brower et al., 1997) or nucleoporin p62 (Fischer et al., 1997) suggest that cnidarians appear to fall very much in the metazoan mainstream. In this study, we found probes for three types of Ca2+ channels: one member of the L-type subfamily and two members of the non-L-type subfamily. This suggests that L-type and non-L-type subfamilies separated before the divergence between diploblastic and triploblastic eukaryotes, at least 700 millions years ago. The L-type S. pistillata a1 subunit Ca2+ channel does not fall into one of the three subtypes (a1C, a1D and a1S) known in mammals. This could be due either to the great evolutionary distance between cnidarians and mammals, or to the existence of an unique form of L-type channel in corals, thus contrasting with that described for example in insects (Pelzer et al., 1989). It can be suggested that the coral a1 subunit Ca2+ channel has evolved from a primitive epithelial Ca2+ channel, which could represent a common ancestor of all Ca2+ channels. Appearance of L-type diversity could have then evolved late in the Bilateria. In this way, the coral Ca2+ channel may then represent a step in Ca2+ channel

evolution whose study may provide important and unique insights into the evolution and physiology of Ca2+ channels.

Acknowledgements This study was conducted as part of the O.O.E. 1996–2000 research program. It was supported by the Council of Europe (Open Partial Agreement on Major Natural and Technological Disasters). We are indebted to Dr V. Paquis and Prof. F. Cuzin for advice, support and use of technical material. We also wish to thank Prof. J.-P. Cuif, Drs M. Bidet and P. Poujeol for fruitful discussions and Prof. M. Lazdunski and Dr Tina Tentori for their comments on the manuscript. The nucleotide sequence reported in this paper has been deposited in the GenBank with accession numbers U64465, AF098943 and AF098944.

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