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J Biol Chem, Vol. 273, Issue 35, 22792-22799, August 28, 1998


Cloning and Functional Expression of a Voltage-gated Calcium Channel alpha 1 Subunit from Jellyfish*

Michael C. Jeziorski, Robert M. Greenberg, Karla S. ClarkDagger , and Peter A. V. Anderson§

From the Whitney Laboratory and the § Departments of Physiology and Neuroscience, University of Florida, St. Augustine, Florida 32086

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha 1 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 alpha 1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha 1, is more similar to vertebrate L-type channels (alpha 1S, alpha 1C, and alpha 1D) than to non-L-type channels (alpha 1A, alpha 1B, and alpha 1E) or low voltage-activated channels (alpha 1G). Expression of CyCaalpha 1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-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+. CyCaalpha 1 thus represents an ancestral L-type alpha 1 subunit with significant functional differences from mammalian L-type channels.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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. 1 and 2). Mammalian Ca2+ channels are composed of a pore-forming alpha 1 subunit and associated beta  and alpha 2delta subunits, as well as a gamma  subunit specific to skeletal muscle. Although the accessory subunits play important roles in channel modulation (3-5), the alpha 1 subunit is primarily responsible for determining the pharmacology and physiology of the resulting current, and heterologous expression of cloned vertebrate alpha 1 subunits has allowed correlation between molecular subtypes and native currents. L-type currents, sensitive to 1,4-dihydropyridines (DHPs),1 are gated by the alpha 1S subtype found in skeletal muscle (6) and the alpha 1C (3) and alpha 1D (7) subtypes expressed in heart, brain, and other tissues. DHP-insensitive, non-L-type alpha 1 subunits include the alpha 1B subtype, which is responsible for the omega -conotoxin GVIA-sensitive N-type current (8), the alpha 1A subtype, which gates the omega -agatoxin IVA-sensitive P/Q-type current (9), and alpha 1E, which gates R-type currents (10). In addition to these high voltage-activated (HVA) alpha 1 subunits, the alpha 1G subunit, responsible for the low voltage-activated (LVA) T-type current, has recently been cloned and characterized (11).

Comparison of the primary structures of the six identified vertebrate HVA alpha 1 subtypes reveals a distinct separation between the L-type and non-L-type channel subfamilies. The similarity among subunits within each class is greater than that between the classes, suggesting that divergence between L-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. 12). Single homologues of both L-type and non-L-type channels have been found in Drosophila (13, 14) and Aplysia (15), and other invertebrate Ca2+ channel sequences (16-18) 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 alpha 1 subunit.

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 (19-21), including the scyphozoan jellyfish Cyanea capillata (22). We report here the cloning and functional expression of CyCaalpha 1, a voltage-gated Ca2+ channel alpha 1 subunit from Cyanea. Although CyCaalpha 1 has the molecular structure of an L-type channel alpha 1 subunit, certain aspects of the pharmacology and physiology of the expressed channel distinguish it from mammalian L-type channels.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of cDNA-- RNA was extracted from Cyanea perirhopalial tissue (consisting of neurons, myoepithelial cells, endoderm, and mesoglea) by homogenization of the tissue in guanidinium isothiocyanate and centrifugation through CsCl. Two degenerate oligonucleotide primers were designed to correspond to highly conserved regions of Ca2+ channel alpha 1 subunit sequences. The sense primer (ATHACNATGGARGGNTGG) corresponds to residues ITMEGW in the pore region of domain I, and the antisense primer (NCCNCCRAAIARYTGCAT, where I = inosine) corresponds to residues MQLFGG in S5 of domain II. cDNA was synthesized from 10 µg of total RNA in a reverse transcription (RT) reaction containing 50 pmol of antisense primer and 200 units of SuperScript II RT (Life Technologies, Inc.). After a 2-h incubation at 42 °C, the RT reaction was heat-inactivated, and 0.5 µl of the cDNA was amplified in a PCR. The PCR, containing 50 pmol of the sense and antisense primers, 200 µM dNTPs, and 2.5 units of Taq DNA polymerase (Fisher), incorporated a touchdown protocol, in which the annealing temperature was decreased from 55 to 45 °C over 11 cycles, then was maintained at 45 °C for 30 cycles. The resulting product was electrophoresed on an agarose gel and purified (Qiaex; Qiagen, Santa Clarita, CA), cloned into pGEM-T (Promega, Madison, WI), and sequenced using the Sequenase 2.0 version (Amersham Pharmacia Biotech) of the dideoxy method. The 5' end of the cDNA was generated by rapid amplification of cDNA ends (Ref. 23), in which a gene-specific antisense primer was used to prime cDNA synthesis from 1 µg of total RNA. The cDNA was tailed with dCTP and terminal transferase (Promega) and then amplified in a PCR with a nested gene-specific primer and an oligo(dG/dI) primer. The 3' end of the open reading frame was determined by isolation of five overlapping fragments. Three of the fragments were generated by PCR from a Cyanea perirhopalial tissue cDNA library, using gene-specific and vector-specific primers, and further amplified with nested primers when necessary. The first fragment isolated in this manner terminated prematurely in a purine-rich region, so a second fragment was generated using inverse PCR, in which Cyanea genomic DNA was digested with HpaII and circularized by ligation, then amplified by PCR to extend the known sequence. Subsequent fragments were generated by PCR from the library and by rapid amplification of cDNA ends.

Once the sequences of all fragments of the cDNA were determined, Cyanea total RNA was used in separate RT reactions to create two additional independent cDNA pools. Overlapping fragments spanning the complete open reading frame of the cDNA were amplified from each cDNA pool and fully sequenced to establish a consensus sequence. A full-length cDNA clone corresponding to the consensus sequence was assembled in a vector (pXENEX1) constructed from portions of two other vectors, pAGA2 (Ref. 24; provided by Maninder Chopra, State University of New York, Buffalo) and pBScMXT (provided by Aguan Wei, Washington University). The multiple cloning site of pXENEX1 is flanked by the 5'-noncoding region of the RNA-4 gene from alfalfa mosaic virus and the 3'-noncoding region of the Xenopus beta -globin gene. The incorporation of a consensus sequence for initiation of translation (25) required that the second amino acid of the open reading frame be changed from Phe to Val.

Northern Blot Analysis-- 100 µg of total RNA from Cyanea perirhopalial tissue was electrophoresed on a glyoxal denaturing gel and blotted to positively charged nylon (Magnagraph; MSI, Westborough, MA). SP6 RNA polymerase was used to synthesize a 32P-labeled antisense riboprobe from a cDNA clone encoding portions of domains I and II of CyCaalpha 1. The blot was incubated with the probe under high stringency hybridization conditions (50% formamide, 5× SSPE, 0.5% SDS, 60 °C) and then was washed three times in 1× SSPE, 0.5% SDS at 65 °C, followed by one wash in 0.1× SSPE, 0.5% SDS at 60 °C.

Expression in Xenopus Oocytes-- The CyCaalpha 1 construct was linearized with NgoMI, purified, and used to transcribe RNA using the T7 version of the mMessage mMachine in vitro transcription kit (Ambion, Austin, TX). Stage V-VI oocytes were removed from Xenopus under MS-222 anesthesia, defolliculated by treatment with 2 mg/ml collagenase in Ca2+-free ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4), and injected with 10-50 ng of RNA, then incubated in sterile ND96 (containing 1.8 mM CaCl2, supplemented with 2.5 mM sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5% horse serum) at 17 °C for 3-10 days. Oocytes were injected with 10-20 nl 100 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (dissolved in 10 mM HEPES) at least 1 h prior to recording to eliminate artifacts caused by the endogenous Ca2+-activated chloride current (26). The standard bath solution consisted of 40 mM Sr(OH)2, 40 mM N-methylglucamine, 10 mM glucose, and 10 mM HEPES, adjusted to pH 7.4 with methanesulfonic acid (27). Where necessary, the Sr(OH)2 in the bath solution was replaced by 40 mM Ca(OH)2 or 40 mM Ba(OH)2. Most drugs were dissolved directly in Sr2+ bath solution and tested by bath perfusion. Nifedipine and isradipine were dissolved in Me2SO and S(-)-BayK 8644 was dissolved in ethanol; then each was diluted in bath medium to a solvent concentration of 1% or less. Similar concentrations of Me2SO or ethanol had no significant effect on the current when administered alone. All DHPs were prepared and applied under subdued light.

Currents were recorded under two-electrode voltage clamp at room temperature (~22 °C). Electrodes were pulled from borosilicate glass and filled with 3 M KCl, resulting in an impedance of 0.1-1 MOmega . Oocytes were clamped at -90 mV (-110 mV in experiments testing inactivation), except as noted. Test protocols were administered using pClamp6 software. Leakage currents were subtracted on-line, using either a P/2 or P/4 protocol of hyperpolarizing prepulses.

Drugs-- The compounds used in these experiments include BAPTA (Molecular Probes, Eugene, OR), nifedipine (Sigma), verapamil hydrochloride (Knoll Pharmaceuticals, Whippany, NJ), isradipine (a generous gift from Dr. Jörg Striessnig, University of Innsbruck), S(-)-BayK 8644 (Research Biochemicals, Inc., Natick, MA), nickel sulfate (Fisher), cadmium sulfate (Fisher), omega -conotoxin GVIA (Calbiochem), omega -conotoxin MVIIC (Calbiochem), and omega -agatoxin IVA (Calbiochem).

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Primary Structure of the Cyanea Ca2+ Channel-- The initial fragment of CyCaalpha 1 was isolated using two degenerate oligonucleotide primers corresponding to regions that are highly conserved in all known Ca2+ channel alpha 1 subunits. These primers were used with RT-PCR to amplify a cDNA segment from Cyanea perirhopalial tissue, and the resulting product was sequenced and found to resemble alpha 1 subunit sequences. The fragment was extended using a series of PCR-based cloning techniques, leading to isolation of a 6857-base pair cDNA, which contains an open reading frame encoding a 1911-amino acid sequence (Fig. 1). The first methionine within the CyCaalpha 1 open reading frame, which is preceded by an in-frame stop codon 36 base pairs upstream, is not surrounded by a consensus sequence for initiation of translation (25), but this motif may not be strongly conserved in cnidarian messages (28). The 3' end of the sequence, which was amplified from an oligo(dT)-primed cDNA library, contains several in-frame stop codons but no consensus polyadenylation signal upstream of the polyadenylated region, suggesting that this tail is spurious. In fact, Northern blot analysis of RNA from Cyanea perirhopalial tissue revealed a single band of approximately 8.5 kb (Fig. 2), indicating that another 1.5-2 kb of the untranslated region(s) has not yet been cloned.


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  		Fig. 1.   CyCaalpha 1 sequence. The deduced amino acid sequence of the Cyanea Ca2+ channel alpha 1 subunit (in boldface; GenBankTM accession number U93075) is aligned with L-type Ca2+ channel alpha 1 subunits from Drosophila (Ref. 13, GenBankTM accession number U00690) and rabbit (Ref. 3, GenBankTM accession number X15539) and non-L-type channels from Drosophila (Ref. 14, GenBankTM accession number U55776) and rat (Ref. 10, GenBankTM accession number L15453). Sequences were aligned using ClustalW (63). Certain regions of the other sequences that exhibit little similarity to the Cyanea sequence are truncated to conserve space; the lengths of deleted regions are indicated in brackets. Residues in the Cyanea sequence that are identical to both of the L-type or both of the non-L-type channel sequences are shaded. The approximate position of transmembrane segments is based upon the alignment. The region involved in interaction with beta  subunits (60) is labeled, and each of the glutamate residues found in the pore regions of all known voltage-gated Ca2+ channel sequences is denoted by a diamond. The putative EF-hand region implicated in Ca2+-dependent inactivation (32, 33) is shown, with matches and mismatches to the consensus EF-hand sequence indicated by + and -, respectively. Residues that have been shown to be involved in dihydropyridine sensitivity of L-type channels (49-53) are indicated by D. Consensus sites for N-linked glycosylation within the CyCaalpha 1 sequence are denoted by g, and potential sites for phosphorylation by cAMP-dependent protein kinase are indicated by p.


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  		Fig. 2.   Northern blot of Cyanea total RNA isolated from perirhopalial tissue. The antisense RNA probe used corresponds to the original PCR product, encoding IS5 to IIS5 of CyCaalpha 1. The estimated size of the band is 8.5 kb.

The alpha 1 subunit of Ca2+ channels comprises four homologous domains arranged symmetrically to form the walls of a pore (6, 29). Each domain consists of six transmembrane segments (S1-S6) and a loop between S5 and S6 that forms part of the ion selectivity pore of the channel. This four-domain structure is evident in CyCaalpha 1. The sequence identity between CyCaalpha 1 and other HVA Ca2+ channel alpha 1 subunits (excluding the weakly conserved amino and carboxyl termini and the intracellular loop between domains II and III) ranges between 49 and 60%. In contrast, the corresponding regions of the LVA alpha 1G subunit from rat (11) are only 34% identical to CyCaalpha 1, and a voltage-gated Na+ channel cloned from Cyanea (28) exhibits only 30% identity. The sequence of CyCaalpha 1 is more similar to those of mammalian L-type alpha 1 subunits (59% average sequence identity in conserved regions) than to those of non-L-type alpha 1 subunits (50% average sequence identity). However, the identity between CyCaalpha 1 and the L-type subunits alpha 1C (60%), alpha 1D (59%), and alpha 1S (58%) does not identify CyCaalpha 1 as a homologue of any mammalian L-type subtype. Similarly, phylogenetic analysis of Ca2+ channel alpha 1 subunit sequences (Fig. 3) places CyCaalpha 1 among the HVA L-type alpha 1 subunits, but suggests that CyCaalpha 1 appeared before the emergence of different subtypes within the L-type family.


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  		Fig. 3.   Phylogenetic analysis of known voltage-gated Ca2+ channel alpha 1 subunit sequences. The regions spanning IS5 to IIS6 and IIIS1 to the conserved region of the carboxyl tail were used for this analysis. Amino acid sequences were aligned using ClustalW. The aligned sequences were used to construct phylogenetic trees using the neighbor-joining method (64), as implemented in PHYLIP version 3.5 (65). The Cyanea sodium channel sequence (GenBankTM accession number L15445) was defined as the outgroup. The tree shown in the figure is identical in branching pattern to a tree produced using a maximum parsimony algorithm (PROTPARS). The branch lengths shown are proportional to calculated phylogenetic distance. GenBankTM accession numbers are as follows (see Fig. 1 for others): rat 1D (M57682), human 1D (M76558), human 1C (Z74996), carp (A37860), rabbit 1S (M23919), human 1S (A55645), Caenorhabditis elegans L (U61951), Musca (S41742), squid (D86600), C. elegans N (U25119), human 1A (X99897), rat 1A (M64373), ray 1B (L12532), human 1B (M94172), rat 1B (M92095), ray 1E (L12531), human 1E (A54972), rat 1G (AF027984).

Most of the highly conserved features of Ca2+ channels appear in CyCaalpha 1. Each pore-forming loop contains a glutamate residue (positions 339, 679, 1085, and 1372) that is present in all known voltage-gated Ca2+ channels and is essential for establishing Ca2+ selectivity (30, 31). The S4 segments, which are involved in voltage sensitivity of Ca2+ channels, display the conserved pattern of positively charged amino acids at every third position. The proximal region of the carboxyl terminus closely matches the consensus sequence for the EF-hand Ca2+-binding motif (32, 33) that plays a role in Ca2+-dependent inactivation of L-type channels (34). However, the region of the I-II interdomain loop that interacts with Ca2+ channel beta  subunits is not highly conserved in the CyCaalpha 1 protein. Of the nine residues found in all other vertebrate and invertebrate HVA alpha 1 subunit sequences (except for one splice variant of Drosophila alpha 1A (14)), only four (Gly-Tyr-Xaa-Xaa-Trp-Ile, beginning at residue 412) are found in CyCaalpha 1. The Gln-Xaa-Xaa-Glu-Arg motif that has been shown in N-type channels to bind the Gbeta gamma subunits of G proteins (35, 36) is also absent from CyCaalpha 1.

The protein contains six consensus cAMP-dependent protein kinase phosphorylation sites within the first two interdomain cytoplasmic loops and the carboxyl terminus. Twenty potential protein kinase C phosphorylation sites are distributed across all three interdomain loops and the amino and carboxyl termini, but no intracellular tyrosine kinase phosphorylation sites appear to be present in the protein. Three consensus N-linked glycosylation sites exist on regions of the protein thought to be exposed to the extracellular side of the membrane, two within the S5-S6 loop of domain I and one within the S1-S2 loop of domain IV.

Functional Characteristics of CyCaalpha 1-- Xenopus oocytes injected with 10-50 ng of CyCaalpha 1 RNA exhibit a large inward current in response to depolarizing test pulses (Fig. 4A). The amplitude of the expressed current in 40 mM Ca2+ or Sr2+ (~1-3 µA) far exceeds that of the native Ca2+ current in oocytes (<50 nA). This robust current was evident in the absence of exogenous beta  or alpha 2delta subunits. The current activates within 2 ms, reaches a peak at 5-10 ms, and then inactivates. The threshold of activation of the current is approximately -20 mV, and maximal current occurs at +15 to +20 mV in 40 mM Sr2+ (Fig. 4B). The value of hinfinity for half-maximal steady-state inactivation of the channel, based on a fit with a Boltzmann function, is -32 mV (Fig. 4C).


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  		Fig. 4.   Expression of CyCaalpha 1. A, an oocyte injected with CyCaalpha 1 RNA was held at -90 mV and tested with a series of 50 ms voltage steps to the indicated potentials, producing the currents shown. The bath contained 40 mM Sr2+. Transient capacitative currents have been deleted for clarity. B, current-voltage relationship of CyCaalpha 1 in the presence of 40 mM Sr2+ (mean ± S.E. of 11 cells). C. Steady-state inactivation of CyCaalpha 1, as determined by 5-s prepulses from a holding potential of -110 mV to the voltages shown, followed by a 50-ms step to 0 mV (n = 7). The fitted Boltzmann curve shown has a slope factor of 8.0 and hinfinity of -32 mV.

In contrast to most other expressed Ca2+ channels, CyCaalpha 1 is less permeable to Ba2+ than to Ca2+, and current is greatest in the presence of Sr2+ (Fig. 5, A and B). In oocytes tested in the presence of 40 mM concentrations of each ion, the ratio of peak current levels for Sr2+:Ca2+:Ba2+ was 1.00:0.73:0.65 (n = 7). As in mammalian Ca2+ channels (37-39), the I-V relationship for Ca2+ permeation is shifted rightward (peak current at +26 mV) compared with that for Ba2+ or Sr2+ (peak current at +14-15 mV), a shift that has been attributed to Ca2+-specific modulation of the gating of the alpha 1 subunit (40). Consequently, the relative permeability of the channel to divalent cations varies with voltage, and the calculated ratio of maximal conductance for Sr2+:Ca2+:Ba2+ is 1.00:0.82:0.66. In the presence of 40 mM Na+ and the absence of divalent cations, peak currents are about 12% those seen in the presence of Sr2+ (data not shown). Due to the larger currents in the presence of Sr2+, all subsequent experiments were conducted with Sr2+ as the charge carrier, except as noted.


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  		Fig. 5.   Ionic selectivity of CyCaalpha 1. A, CyCaalpha 1 current produced in a single cell by a 50-ms voltage step to +20 mV in the presence of 40 mM concentrations of the cations indicated. Ions in this experiment were presented in the order Ca2+, Ba2+, Sr2+; a subsequent recording in Ca2+ bath solution indicated no current rundown. B, permeability of CyCaalpha 1 to Ba2+ (squares), Ca2+ (triangles), and Sr2+ (circles). Each cell was tested in the presence of all three ions (n = 7). The order in which bath solutions were applied was varied for each experiment, and the first bath solution tested was reapplied after all bath changes were complete to ensure that no current rundown had occurred. The data points were fitted by the Boltzmann function I = gmax(V - Vrev)/(1 + exp(-(V - V1/2)/k), where I is whole cell current at test potential V; gmax is maximal conductance; Vrev is reversal potential; V1/2 is potential for half-activation; and k is slope factor. The parameter values for Sr2+, Ba2+, and Ca2+, respectively, are gmax = 101, 67, and 83 µS; Vrev = 69, 67, and 74 mV; V1/2 = 2.4, 0.6, and 12.8 mV; and k = 6.7, 7.5, and 7.6 mV. C, time constant of current inactivation in the presence of each permeant ion, expressed as a function of voltage. Single exponentials were fitted to the decaying phase of the currents measured in B. Symbols are as indicated in B.

As illustrated in Fig. 5A, the CyCaalpha 1 current inactivates in the presence of all three divalent cations. Although the rate of current decay varies slightly with voltage, Ba2+ currents inactivate with a time constant between 30 and 36 ms at test potentials above +10 mV, and Sr2+ currents with a time constant between 27 and 30 ms (Fig. 5C). The rate of inactivation is increased when Ca2+ is the permeant ion, indicating that CyCaalpha 1 exhibits the Ca2+-dependent inactivation characteristic of mammalian L-type Ca2+ channels (34).

Pharmacology of CyCaalpha 1-- The CyCaalpha 1 current is surprisingly insensitive to DHPs. Whereas most mammalian L-type channels are fully blocked by 10 µM nifedipine, only 50% inhibition of the CyCaalpha 1 current is produced by 100 µM nifedipine (Fig. 6A). Similarly, the potent DHP isradipine produces only 36% inhibition at a concentration of 100 µM. The potency of nifedipine or isradipine block is not enhanced by raising the holding potential from -90 mV to -60 mV or by coexpressing an exogenous Ca2+ channel beta  subunit (data not shown). CyCaalpha 1 is similarly insensitive to the phenylalkylamine verapamil; a 100 µM concentration inhibits the current by 42%.


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  		Fig. 6.   Pharmacology of CyCaalpha 1. A, inhibition of the CyCaalpha 1 Sr2+ current by varying concentrations of the DHP antagonist nifedipine (open circles; n = 5) and the DHP agonist S(-)-BayK 8644 (open squares; n = 4). The inhibition produced by 100 µM concentrations of the DHP antagonist isradipine (solid triangle; n = 5) and the phenylalkylamine verapamil (solid inverted triangle; n = 3) are also shown. Data points reflect peak current elicited by a 250-ms voltage step to +20 mV from a holding potential of -90 mV. B, Block of the CyCaalpha 1 Sr2+ current by Cd2+ (circles; n = 6) or Ni2+ (squares; n = 6). Recording conditions are as described in A.

S(-)-BayK 8644, a DHP that acts as an agonist on vertebrate L-type channels, also has an unexpected action on the CyCaalpha 1 current. The current is not potentiated by concentrations of S(-)-BayK 8644 ranging from 1 to 100 µM (Fig. 6A). Instead, S(-)-BayK 8644 inhibits the current in a manner comparable to that of nifedipine.

The N-type channel blocker omega -conotoxin GVIA (10 µM) and the P/Q-type channel ligands omega -conotoxin MVIIC (5 µM) and omega -agatoxin IVA (1 µM) have no effect on the CyCaalpha 1 current (data not shown). In addition, CyCaalpha 1 is potently blocked by Cd2+ (IC50 = 2.1 µM; Fig. 6B), but, in contrast to R-type or T-type channels, is not highly sensitive to Ni2+ block (IC50 = 570 µM).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

CyCaalpha 1 Represents an Early L-type Ca2+ Channel-- Cnidarians, the earliest existing metazoans, appeared 600 million years before the emergence of mammals (41). Voltage-gated Ca2+ currents have been identified in more ancient eukaryotes, such as protists (42-44) and plants (45), but cnidarians are the simplest organisms in which movement is governed by a neuromuscular system. Although the tissue distribution of CyCaalpha 1 is unknown, the channel is likely to be a component of Cyanea neural or muscle cells. The perirhopalial tissue from which the CyCaalpha 1 cDNA was derived contains several cell types, including neurons, muscle, ectodermal and endodermal epithelial cells, and cnidocytes. Ca2+ currents in Cyanea muscle cells have not yet been characterized, but Cyanea neurons possess a high voltage-activated Ca2+ current of undetermined type (22). Scyphozoan jellyfish do not have electrically excitable epithelial cells, however, and no inward currents can be recorded from their cnidocytes (46). This evidence suggests that CyCaalpha 1 represents one of the earliest examples of a neuromuscular voltage-gated Ca2+ channel.

Sequence analysis divides all previously known HVA Ca2+ channel alpha 1 subunits into two classes, termed L-type and non-L-type. Although the CyCaalpha 1 sequence varies significantly from those of mammalian channels, it clearly shares more amino acid identity with L-type channels than with non-L-type channels. An alpha 1 subunit that appeared before the divergence of the two HVA channel subfamilies would be expected to resemble members of both classes equally, as is seen with the LVA alpha 1G subunit. The identification of CyCaalpha 1 as an L-type channel implies that the separation of HVA Ca2+ channels into L-type and non-L-type classes occurred prior to or concurrent with the evolution of neuromuscular systems. Non-L-type channel genes have been identified in nematodes (17) and platyhelminths,2 but we have not yet obtained molecular evidence for the existence of non-L-type Ca2+ channels in Cyanea.

CyCaalpha 1 does not preferentially resemble any one of the mammalian subtypes (alpha 1S, alpha 1C, and alpha 1D) of the L-type class. Instead, phylogenetic analysis indicates that CyCaalpha 1, and perhaps other invertebrate L-type channels, represents an ancestral form that emerged before the appearance of the different mammalian L-type subtypes. Therefore, CyCaalpha 1 has not been assigned a subscript to conform to the accepted nomenclature for vertebrate Ca2+ channels. Although subtype designations have been assigned to certain invertebrate Ca2+ channels on the basis of sequence similarity, multiple molecular subtypes within the L-type or non-L-type class have thus far been identified only in vertebrates (e.g. Ref. 47).

The primary structure of CyCaalpha 1 also relates to evolution within the voltage-gated ion channel superfamily. The pore-forming subunit of voltage-gated Na+ channels, which has a structure homologous to that of the Ca2+ channel alpha 1 subunit, is thought to have arisen from Ca2+ channels at a time roughly coinciding with the appearance of metazoans (48). However, CyCaalpha 1 exhibits no greater similarity to a Cyanea Na+ channel (28) than do mammalian Ca2+ channel alpha 1 subunits; thus, Na+ channels and Ca2+ channels are already highly divergent at the level of cnidarians.

The CyCaalpha 1 Current Differs from Vertebrate L-type Currents-- The molecular structure of CyCaalpha 1 clearly identifies it as L-type, but the pharmacological response of the expressed current differs considerably from that of mammalian L-type currents. Although CyCaalpha 1 is unaffected by peptide toxins specific for non-L-type channels and is only weakly sensitive to Ni2+, which potently blocks T-type and R-type channels, the CyCaalpha 1 current is also quite insensitive to block by DHPs. Significant current remains in the presence of 10 µM nifedipine, a concentration sufficient to completely block the rabbit alpha 1C channel expressed in oocytes (3), and full inhibition of the CyCaalpha 1 current is not evident below 1 mM nifedipine. The DHP isradipine and the phenylalkylamine verapamil are similarly ineffective in blocking the current. Furthermore, S(-)-BayK 8644, a DHP enantiomer that potentiates vertebrate L-type currents, produces only weak inhibition of the CyCaalpha 1 current. In fact, the low potency of the antagonist and agonist actions, as well as the shallowness of the concentration-response curves, indicates that the moderate inhibition seen at higher drug concentrations may be due to nonspecific effects.

The insensitivity of CyCaalpha 1 to DHPs implies that the structure of the DHP-binding pocket identified in higher L-type channels is altered in CyCaalpha 1. The residues throughout domains III and IV that have been implicated in conferring DHP sensitivity to L-type channels (49-53) are identical between CyCaalpha 1 and alpha 1C in all but two positions. The substitution of Gly-903 in CyCaalpha 1 in place of Phe may not be critical; an analogous mutation in the human alpha 1C channel has little effect on DHP block (49). More significant is the presence of Ile-1128. The Met residue at the corresponding position in the alpha 1C subunit influences both block and potentiation by DHPs (52-54). The Musca and Drosophila L-type alpha 1 subunits also contain a hydrophobic substitution for Met at this site; in fact, the replacement of Met-1188 (alpha 1C numbering) is responsible for the decreased response of Musca-alpha 1C chimeric channels to DHPs (52). The experiments presented herein directly show DHP insensitivity in an invertebrate L-type alpha 1 subunit, which is in accord with reports that Ca2+ currents in lower invertebrates exhibit decreased sensitivity to DHP block. The Ca2+ current in Paramecium is unaffected by DHPs (43), and neuronal Ca2+ currents in the hydrozoan jellyfish Polyorchis (21) and the flatworm Bdelloura (55) are only partially blocked by 10-100 µM nifedipine (although these Ca2+ currents may not be gated by L-type channel homologues). Molluscs are the earliest organisms in which potent block of Ca2+ currents by DHPs has been reported (56), although the current is unaffected by racemic BayK 8644. The present results demonstrate that the actions of DHPs, which are generally used to define a current as L-type, may not reliably identify L-type channel homologues in lower organisms.

The CyCaalpha 1 subunit also exhibits a distinctive pattern of whole-cell permeability to divalent cations. Replacement of Ca2+ by Ba2+ results in decreased current in oocytes expressing CyCaalpha 1, and Sr2+ produces larger currents than Ca2+. This selectivity profile (ISr > ICa > IBa) is distinct from that of most expressed vertebrate alpha 1 subunits (IBa > ISr > ICa). However, the permeability of the non-L-type rat alpha 1E subunit in oocytes (39), which differs even from that of other mammalian alpha 1E subunits (38), is similar to that of CyCaalpha 1. Inferring channel permeability from whole-cell recordings is made difficult by effects of divalent cations on the mean open time of the channel (57), and patch clamp experiments are necessary to accurately determine permeability. Nevertheless, the similar whole-cell profiles of the Cyanea and rat alpha 1E channels indicate that they may share structural features distinct from those of most known vertebrate HVA Ca2+ channels. Alignment of the pore region sequences of CyCaalpha 1 and mammalian alpha 1 subunits reveals no single amino acid substitution unique to CyCaalpha 1 and rat alpha 1E. An aspartate residue found in the domain I pore region of most alpha 1 subunits is replaced in rat alpha 1E by a threonine (Thr-264), which has been proposed to play a role in altered ionic selectivity (39). However, the aspartate is conserved in CyCaalpha 1 (Asp-343).

One region of CyCaalpha 1 that is poorly conserved is the domain in the I-II intracellular loop that interacts with Ca2+ channel beta  subunits. The beta  subunit alters the activation and inactivation kinetics of the alpha 1 subunit and increases current amplitude (5, 58), and is also involved in trafficking of the alpha 1 subunit to the plasma membrane (59). The I-II loop of the alpha 1 subunit contains an 18-amino acid cassette to which the beta  subunit binds (60, 61). Nine residues are conserved within this cassette in all known vertebrate Ca2+ channel sequences (QQXEXXLXGYXXWIXXXE), but only four are found in CyCaalpha 1. Of these four, three (Tyr-413, Trp-416, and Ile-417) have been shown to be the most critical residues for beta  subunit binding (62). Although CyCaalpha 1 does not require an exogenous beta  subunit for functional expression, we have cloned a beta  subunit from Cyanea that is able to modulate the activity of CyCaalpha 1.3

These findings illustrate the value of studying a phylogenetically distant voltage-gated Ca2+ channel. CyCaalpha 1 diverged from the ancestor of vertebrate Ca2+ channels over half a billion years ago, creating the opportunity for variation in all but the most structurally constrained regions of the protein. The ability to correlate this novel structural information with the physiology of the expressed current provides a new avenue for examining Ca2+ channel function.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported in part by National Science Foundation Grant IBN-9410565 (to P. A. V. A.), University of Florida Division of Sponsored Research Grant 94022315 (to R. M. G.), and National Research Service Award MH 10625 (to M. C. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U93075.

Dagger Supported by a Research Experience for Undergraduates Grant IBN-9222803 from the National Science Foundation.

To whom correspondence should be addressed: the Whitney Laboratory, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32086. Tel.: 904-461-4028; Fax: 904-461-4008; E-mail: paa{at}whitney.ufl.edu.

The abbreviations used are: DHP, dihydropyridine; HVA, high voltage-activated; LVA, low voltage-activated; PCR, polymerase chain reaction; RT, reverse transcription; kb, kilobase pairs; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

2 R. Greenberg, unpublished observations.

3 M. C. Jeziorski, unpublished observations.

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Top
Abstract
Introduction
Procedures
Results
Discussion
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