Title: Cloning and Functional Expression of a Voltage-gated Calcium Channel α1 Subunit from Jellyfish
Abstract: Voltage-gated Ca2+channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel α1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the α1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfishCyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaα1, is more similar to vertebrateL-type channels (α1S, α1C, and α1D) than to non-L-type channels (α1A, α1B, and α1E) or low voltage-activated channels (α1G). Expression of CyCaα1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrateL-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(−)-BayK 8644. In addition, the channel is less permeable to Ba2+than to Ca2+ and is more permeable to Sr2+. CyCaα1 thus represents an ancestral L-type α1 subunit with significant functional differences from mammalian L-type channels. Voltage-gated Ca2+channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel α1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the α1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfishCyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaα1, is more similar to vertebrateL-type channels (α1S, α1C, and α1D) than to non-L-type channels (α1A, α1B, and α1E) or low voltage-activated channels (α1G). Expression of CyCaα1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrateL-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(−)-BayK 8644. In addition, the channel is less permeable to Ba2+than to Ca2+ and is more permeable to Sr2+. CyCaα1 thus represents an ancestral L-type α1 subunit with significant functional differences from mammalian L-type channels. Voltage-gated Ca2+ channels are essential for coupling the depolarization of excitable cells to Ca2+ influx across the plasma membrane, leading to initiation of second messenger cascades and other intracellular events. The various voltage-gated Ca2+ currents that have been described in vertebrate and invertebrate tissues were originally defined by their biophysical and pharmacological differences. Isolation of cDNAs encoding several mammalian Ca2+ channel subtypes has provided a molecular basis for the diversity of Ca2+ currents in higher vertebrates (for review see Refs. 1Perez-Reyes E. Schneider T. Kidney Int. 1995; 48: 1111-1124Abstract Full Text PDF PubMed Scopus (148) Google Scholar and 2Stea A. Soong T.W. Snutch T.P. North R.A. Handbook of Receptors and Channels: Ligand- and Voltage-gated Ion Channels. CRC Press, Inc., Boca Raton, FL1995: 113-151Google Scholar). Mammalian Ca2+channels are composed of a pore-forming α1 subunit and associated β and α2δ subunits, as well as a γ subunit specific to skeletal muscle. Although the accessory subunits play important roles in channel modulation (3Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takashima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (762) Google Scholar, 4Lacerda A.E. Kim H.S. Ruth P. Perez-Reyes E. Flockerzi V. Hofmann F. Birnbaumer L. Brown A.M. Nature. 1991; 352: 527-530Crossref PubMed Scopus (240) Google Scholar, 5Varadi G. Lory P. Schultz D. Varadi M. Schwartz A. Nature. 1991; 352: 159-162Crossref PubMed Scopus (229) Google Scholar), the α1subunit is primarily responsible for determining the pharmacology and physiology of the resulting current, and heterologous expression of cloned vertebrate α1 subunits has allowed correlation between molecular subtypes and native currents. L-type currents, sensitive to 1,4-dihydropyridines (DHPs), 1The abbreviations used are: DHPdihydropyridineHVAhigh voltage-activatedLVAlow voltage-activatedPCRpolymerase chain reactionRTreverse transcriptionkbkilobase pairsBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid.1The abbreviations used are: DHPdihydropyridineHVAhigh voltage-activatedLVAlow voltage-activatedPCRpolymerase chain reactionRTreverse transcriptionkbkilobase pairsBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. are gated by the α1S subtype found in skeletal muscle (6Tanabe T. Takeshima H. Mikami A. Flockerzi V. Takahashi H. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1987; 328: 313-318Crossref PubMed Scopus (956) Google Scholar) and the α1C (3Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takashima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (762) Google Scholar) and α1D (7Williams M.E. Feldman D.H. McCue A.F. Brenner R. Veliçelebi G. Ellis S.B. Harpold M.M. Neuron. 1992; 8: 71-84Abstract Full Text PDF PubMed Scopus (437) Google Scholar) subtypes expressed in heart, brain, and other tissues. DHP-insensitive, non-L-type α1 subunits include the α1B subtype, which is responsible for the ω-conotoxin GVIA-sensitive N-type current (8Williams M.E. Brust P.F. Feldman D.H. Patthi S. Simerson S. Maroufi A. McCue A.F. Veliçelebi G. Ellis S.B. Harpold M.M. Science. 1992; 257: 389-395Crossref PubMed Scopus (415) Google Scholar), the α1A subtype, which gates the ω-agatoxin IVA-sensitive P/Q-type current (9Mori Y. Friedrich T. Kim M.-S. Mikami A. Nakai J. Ruth P. Bosse E. Hofmann F. Flockerzi V. Furuichi T. Mikoshiba K. Imoto K. Tanabe T. Numa S. Nature. 1991; 350: 398-402Crossref PubMed Scopus (704) Google Scholar), and α1E, which gates R-type currents (10Soong T.W. Stea A. Hodson C.D. Dubel S.J. Vincent S.R. Snutch T.P. Science. 1993; 260: 1133-1136Crossref PubMed Scopus (436) Google Scholar). In addition to these high voltage-activated (HVA) α1 subunits, the α1G subunit, responsible for the low voltage-activated (LVA) T-type current, has recently been cloned and characterized (11Perez-Reyes E. Cribbs L.L. Daud A. Lacerda A.E. Barclay J. Williamson M.P. Fox M. Rees M. Lee J.-H. Nature. 1998; 391: 896-900Crossref PubMed Scopus (636) Google Scholar).Comparison of the primary structures of the six identified vertebrate HVA α1 subtypes reveals a distinct separation between theL-type and non-L-type channel subfamilies. The similarity among subunits within each class is greater than that between the classes, suggesting that divergence betweenL-type and non-L-type channels constituted the first step in the evolution of known HVA Ca2+ channels. This division between channel classes is maintained in the invertebrate Ca2+ channels that have been cloned (for review see Ref.12Skeer J.M. Norman R.I. Sattelle D.B. Biol. Rev. 1996; 71: 137-154Crossref Scopus (16) Google Scholar). Single homologues of both L-type and non-L-type channels have been found inDrosophila (13Zheng W. Feng G. Ren D. Eberl D.F. Hannan F. Dubald M. Hall L.M. J. Neurosci. 1995; 15: 1132-1143Crossref PubMed Google Scholar, 14Smith L.A. Wang X. Peixoto A.A. Neumann E.K. Hall L.M. Hall J.C. J. Neurosci. 1996; 16: 7868-7879Crossref PubMed Google Scholar) and Aplysia (15White B.H. Kaczmarek L.K. J. Neurosci. 1997; 17: 1582-1595Crossref PubMed Google Scholar), and other invertebrate Ca2+ channel sequences (16Grabner M. Bachmann A. Rosenthal F. Striessnig J. Schulz C. Tautz D. Glossmann H. FEBS Lett. 1994; 339: 189-194Crossref PubMed Scopus (29) Google Scholar, 17Schafer W.R. Kenyon C.J. Nature. 1995; 375: 73-78Crossref PubMed Scopus (227) Google Scholar, 18Kimura T. Shouno O. Hirota K. Saito T. Matsumoto G. Sato C. Biochem. Biophys. Res. Commun. 1997; 230: 147-154Crossref PubMed Scopus (8) Google Scholar) exhibit clear resemblance to one of the two channel subfamilies. However, a thorough understanding of the relationship between invertebrate Ca2+channel structure and physiology requires functional expression of a cloned invertebrate Ca2+ channel α1subunit.Cnidarians, which include jellyfish, anemones, and corals, are the earliest existing organisms to possess a neuromuscular system. Voltage-gated Ca2+ currents have been recorded from neural and muscle cells of several cnidarian species (19Mackie G.O. Meech R.W. Nature. 1985; 313: 791-793Crossref PubMed Scopus (51) Google Scholar, 20Holman M.A. Anderson P.A.V. J. Exp. Biol. 1991; 161: 333-346Google Scholar, 21Przysiezniak J. Spencer A.N. J. Neurosci. 1992; 12: 2065-2078Crossref PubMed Google Scholar), including the scyphozoan jellyfish Cyanea capillata (22Anderson P.A.V. J. Exp. Biol. 1987; 133: 231-248Google Scholar). We report here the cloning and functional expression of CyCaα1, a voltage-gated Ca2+ channel α1 subunit fromCyanea. Although CyCaα1 has the molecular structure of an L-type channel α1 subunit, certain aspects of the pharmacology and physiology of the expressed channel distinguish it from mammalian L-type channels. Voltage-gated Ca2+ channels are essential for coupling the depolarization of excitable cells to Ca2+ influx across the plasma membrane, leading to initiation of second messenger cascades and other intracellular events. The various voltage-gated Ca2+ currents that have been described in vertebrate and invertebrate tissues were originally defined by their biophysical and pharmacological differences. Isolation of cDNAs encoding several mammalian Ca2+ channel subtypes has provided a molecular basis for the diversity of Ca2+ currents in higher vertebrates (for review see Refs. 1Perez-Reyes E. Schneider T. Kidney Int. 1995; 48: 1111-1124Abstract Full Text PDF PubMed Scopus (148) Google Scholar and 2Stea A. Soong T.W. Snutch T.P. North R.A. Handbook of Receptors and Channels: Ligand- and Voltage-gated Ion Channels. CRC Press, Inc., Boca Raton, FL1995: 113-151Google Scholar). Mammalian Ca2+channels are composed of a pore-forming α1 subunit and associated β and α2δ subunits, as well as a γ subunit specific to skeletal muscle. Although the accessory subunits play important roles in channel modulation (3Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takashima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (762) Google Scholar, 4Lacerda A.E. Kim H.S. Ruth P. Perez-Reyes E. Flockerzi V. Hofmann F. Birnbaumer L. Brown A.M. Nature. 1991; 352: 527-530Crossref PubMed Scopus (240) Google Scholar, 5Varadi G. Lory P. Schultz D. Varadi M. Schwartz A. Nature. 1991; 352: 159-162Crossref PubMed Scopus (229) Google Scholar), the α1subunit is primarily responsible for determining the pharmacology and physiology of the resulting current, and heterologous expression of cloned vertebrate α1 subunits has allowed correlation between molecular subtypes and native currents. L-type currents, sensitive to 1,4-dihydropyridines (DHPs), 1The abbreviations used are: DHPdihydropyridineHVAhigh voltage-activatedLVAlow voltage-activatedPCRpolymerase chain reactionRTreverse transcriptionkbkilobase pairsBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid.1The abbreviations used are: DHPdihydropyridineHVAhigh voltage-activatedLVAlow voltage-activatedPCRpolymerase chain reactionRTreverse transcriptionkbkilobase pairsBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. are gated by the α1S subtype found in skeletal muscle (6Tanabe T. Takeshima H. Mikami A. Flockerzi V. Takahashi H. Kangawa K. Kojima M. Matsuo H. Hirose T. Numa S. Nature. 1987; 328: 313-318Crossref PubMed Scopus (956) Google Scholar) and the α1C (3Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takashima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (762) Google Scholar) and α1D (7Williams M.E. Feldman D.H. McCue A.F. Brenner R. Veliçelebi G. Ellis S.B. Harpold M.M. Neuron. 1992; 8: 71-84Abstract Full Text PDF PubMed Scopus (437) Google Scholar) subtypes expressed in heart, brain, and other tissues. DHP-insensitive, non-L-type α1 subunits include the α1B subtype, which is responsible for the ω-conotoxin GVIA-sensitive N-type current (8Williams M.E. Brust P.F. Feldman D.H. Patthi S. Simerson S. Maroufi A. McCue A.F. Veliçelebi G. Ellis S.B. Harpold M.M. Science. 1992; 257: 389-395Crossref PubMed Scopus (415) Google Scholar), the α1A subtype, which gates the ω-agatoxin IVA-sensitive P/Q-type current (9Mori Y. Friedrich T. Kim M.-S. Mikami A. Nakai J. Ruth P. Bosse E. Hofmann F. Flockerzi V. Furuichi T. Mikoshiba K. Imoto K. Tanabe T. Numa S. Nature. 1991; 350: 398-402Crossref PubMed Scopus (704) Google Scholar), and α1E, which gates R-type currents (10Soong T.W. Stea A. Hodson C.D. Dubel S.J. Vincent S.R. Snutch T.P. Science. 1993; 260: 1133-1136Crossref PubMed Scopus (436) Google Scholar). In addition to these high voltage-activated (HVA) α1 subunits, the α1G subunit, responsible for the low voltage-activated (LVA) T-type current, has recently been cloned and characterized (11Perez-Reyes E. Cribbs L.L. Daud A. Lacerda A.E. Barclay J. Williamson M.P. Fox M. Rees M. Lee J.-H. Nature. 1998; 391: 896-900Crossref PubMed Scopus (636) Google Scholar). dihydropyridine high voltage-activated low voltage-activated polymerase chain reaction reverse transcription kilobase pairs 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. dihydropyridine high voltage-activated low voltage-activated polymerase chain reaction reverse transcription kilobase pairs 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Comparison of the primary structures of the six identified vertebrate HVA α1 subtypes reveals a distinct separation between theL-type and non-L-type channel subfamilies. The similarity among subunits within each class is greater than that between the classes, suggesting that divergence betweenL-type and non-L-type channels constituted the first step in the evolution of known HVA Ca2+ channels. This division between channel classes is maintained in the invertebrate Ca2+ channels that have been cloned (for review see Ref.12Skeer J.M. Norman R.I. Sattelle D.B. Biol. Rev. 1996; 71: 137-154Crossref Scopus (16) Google Scholar). Single homologues of both L-type and non-L-type channels have been found inDrosophila (13Zheng W. Feng G. Ren D. Eberl D.F. Hannan F. Dubald M. Hall L.M. J. Neurosci. 1995; 15: 1132-1143Crossref PubMed Google Scholar, 14Smith L.A. Wang X. Peixoto A.A. Neumann E.K. Hall L.M. Hall J.C. J. Neurosci. 1996; 16: 7868-7879Crossref PubMed Google Scholar) and Aplysia (15White B.H. Kaczmarek L.K. J. Neurosci. 1997; 17: 1582-1595Crossref PubMed Google Scholar), and other invertebrate Ca2+ channel sequences (16Grabner M. Bachmann A. Rosenthal F. Striessnig J. Schulz C. Tautz D. Glossmann H. FEBS Lett. 1994; 339: 189-194Crossref PubMed Scopus (29) Google Scholar, 17Schafer W.R. Kenyon C.J. Nature. 1995; 375: 73-78Crossref PubMed Scopus (227) Google Scholar, 18Kimura T. Shouno O. Hirota K. Saito T. Matsumoto G. Sato C. Biochem. Biophys. Res. Commun. 1997; 230: 147-154Crossref PubMed Scopus (8) Google Scholar) exhibit clear resemblance to one of the two channel subfamilies. However, a thorough understanding of the relationship between invertebrate Ca2+channel structure and physiology requires functional expression of a cloned invertebrate Ca2+ channel α1subunit. Cnidarians, which include jellyfish, anemones, and corals, are the earliest existing organisms to possess a neuromuscular system. Voltage-gated Ca2+ currents have been recorded from neural and muscle cells of several cnidarian species (19Mackie G.O. Meech R.W. Nature. 1985; 313: 791-793Crossref PubMed Scopus (51) Google Scholar, 20Holman M.A. Anderson P.A.V. J. Exp. Biol. 1991; 161: 333-346Google Scholar, 21Przysiezniak J. Spencer A.N. J. Neurosci. 1992; 12: 2065-2078Crossref PubMed Google Scholar), including the scyphozoan jellyfish Cyanea capillata (22Anderson P.A.V. J. Exp. Biol. 1987; 133: 231-248Google Scholar). We report here the cloning and functional expression of CyCaα1, a voltage-gated Ca2+ channel α1 subunit fromCyanea. Although CyCaα1 has the molecular structure of an L-type channel α1 subunit, certain aspects of the pharmacology and physiology of the expressed channel distinguish it from mammalian L-type channels. We are grateful to Dr. Aguan Wei and Dr. Maninder Chopra for their donation of plasmid vectors; Dr. Edward Perez-Reyes for supplying mammalian calcium channel cDNAs; and Drs. Jörg Mitterdorfer, Jörg Striessnig, Edward Perez-Reyes, and William Harvey for helpful comments on the manuscript.