The α1 Subunit EGL-19, the α2/δ Subunit UNC-36, and the β Subunit CCB-1 Underlie Voltage-dependent Calcium Currents in Caenorhabditis elegans Striated Muscle*

Background: The composition of voltage-gated calcium channels is important for function. Results: In C. elegans, calcium currents are altered in mutants of one main subunit (EGL-19) or the accessory subunits UNC-36 and CCB-1. Conclusion: Voltage-gated calcium channels of C. elegans striated muscles are composed of EGL-19, UNC-36, and CCB-1. Significance: C. elegans could be a new model to study the roles of accessory subunits. Voltage-gated calcium channels, which play key roles in many physiological processes, are composed of a pore-forming α1 subunit associated with up to three auxiliary subunits. In vertebrates, the role of auxiliary subunits has mostly been studied in heterologous systems, mainly because of the severe phenotypes of knock-out animals. The genetic model Caenorhabditis elegans has all main types of voltage-gated calcium channels and strong loss-of-function mutations in all pore-forming and auxiliary subunits; it is therefore a useful model to investigate the roles of auxiliary subunits in their native context. By recording calcium currents from channel and auxiliary subunit mutants, we molecularly dissected the voltage-dependent calcium currents in striated muscle of C. elegans. We show that EGL-19 is the only α1 subunit that carries calcium currents in muscle cells. We then demonstrate that the α2/δ subunit UNC-36 modulates the voltage dependence, the activation kinetics, and the conductance of calcium currents, whereas another α2/δ subunit TAG-180 has no effect. Finally, we characterize mutants of the two β subunits, CCB-1 and CCB-2. CCB-1 is necessary for viability, and voltage-dependent calcium currents are abolished in the absence of CCB-1 whereas CCB-2 does not affect currents. Altogether these results show that EGL-19, UNC-36, and CCB-1 underlie voltage-dependent calcium currents in C. elegans striated muscle.

calcium, such as muscle contraction, neurotransmitter release, and gene regulation. They are composed of a pore-forming ␣1 subunit associated with up to three auxiliary subunits, ␣2/␦, ␤, and ␥ (1). Besides the ion channel pore, the ␣1 subunit contains the voltage sensor, the selectivity filter, and binding sites for several drugs. Auxiliary subunits, in particular ␤ and ␣2/␦, modulate biophysical properties, influence regulation of the channels by other proteins and molecules, and play a crucial role in trafficking (2,3). However, the roles of these subunits have mostly been studied in heterologous systems; these interactions may therefore not correspond to real subunit interactions in their native context. In vertebrates, double mutants of ␤ and ␣2/␦ subunits have never been generated, mainly because most knock-out animals are very sick (2,3). The use of genetic models, such as Caenorhabditis elegans, Drosophila melanogaster, or Danio rerio, could be a fruitful approach to understand the in situ roles of auxiliary subunits in muscle cells. However, the auxiliary subunits in these organisms have only been investigated by a few studies which did not fully characterize their native roles (4 -11).
Voltage-dependent calcium currents have been recorded in neurons and muscle cells from the pharynx and the body wall (15,17,18); however, molecular dissection of voltage-dependent calcium currents is still incomplete in this organism. In situ characterization of voltage-dependent calcium currents has shown that the ␣1 subunit CCA-1 underlies a T-type cur-rent in pharyngeal muscle whereas EGL-19 carries L-type currents both in pharyngeal and body muscles (15,19,20). Yet, it is still unclear whether other ␣1 subunits contribute to calcium currents in these tissues. The role of auxiliary subunits in C. elegans is even less well defined. Indirect calcium imaging experiments have revealed that UNC-36 influences calcium transients in pharyngeal muscles and in sensory neurons (5,9,21). However, the direct effects of UNC-36 on voltage-dependent calcium currents have not been determined by electrophysiological recording. Until now, no functional data have been published on ␤ subunits in C. elegans.
In this study, we molecularly dissect the voltage-dependent calcium current in C. elegans striated muscle. By recording voltage-dependent calcium currents from body muscle cells of wild-type and mutant animals, we demonstrate that EGL-19 is the only ␣1 subunit that carries current in these cells. In addition, we identify auxiliary subunits that regulate EGL-19: we show that the ␣2/␦ subunit UNC-36 and the ␤ subunit CCB-1 affect voltage dependence, kinetics, and conductance of voltage-dependent calcium currents.

EXPERIMENTAL PROCEDURES
Strains-All C. elegans strains were grown at 20°C according to Ref. 22 unc-2(mu74) and unc-2(lj1) are deletions leading to truncated proteins in the mid fourth and third domain, respectively (23). cca-1(ad1650) is a deletion leading to a truncated protein in the mid second domain (24). unc-36(e251) is a nonsense mutation leading to a truncated protein in the first third (25). The nca-2(gk5); nca-1(gk9), ccb-1(gk18), tag-180(ok779), and ccb-2(ok862) strains carry large mutations (see WormBase on the Internet) likely resulting in nonfunctional proteins. unc-36(ad698) mutation was identified by L. Lobel and W. Schafer, 3 and we verified its localization by sequencing: we found a C to T mutation leading to an early R412stop codon (WormBase release WS224).
Because all lines showed the same GFP expression pattern, only one was analyzed using confocal microscopy.
A transcriptional ccb-2 promoter-driven GFP construct was made using 5.9 kb of ccb-2 5Ј upstream sequences. The fragment was amplified from genomic DNA using oligonucleotides 5Ј-GCGCGCATGCCGTCCAAACTGTGAGGG-3Ј and 5Ј-GCGCCTGCAGCCTTTCTCGTCAAGACTCCCATC-3Ј, that append PstI and SphI restriction sites, respectively. The promoter fragment was inserted into a PstI-and SphI-digested Fire laboratory vector pPD95.75. The plasmid was injected at 5 ng/ml using Punc-122:GFP as a co-injection marker. Three transgenic lines were generated. Because all lines showed the same GFP expression pattern, only one was analyzed using confocal microscopy.
A muscle rescue construct (pCFJ262) was made in a threeway Gateway reaction by reacting Pmyo-3, unc-36 cDNA, and an unc-54 utr and verified by digestion. unc-36(e251) animals were rescued in the body muscles by injection of 10 ng/l pCFJ262, 20 ng/l Pmyo-3:GFP, and 1-kb ladder as stuffer.
Fluorescence Microscopy-Young adults carrying the GFP transcriptional constructs were transferred into a drop of M9 containing 20 mM sodium azide on a top of an agarose pad. Animals were imaged on an LSM 5 Exciter laser scanning confocal microscope (Zeiss) equipped with a ϫ63 oil immersion objective ϫ1.4 numerical aperture.
Behavioral Test-Ten to 20 L4 larvae were placed on freshly seeded NGM plates at 15°C. The following day, individual worms were transferred into a microtiter well (Corning) containing 50 l of M9 medium on top of 2% agar in M9. The worms were allowed to recover from the transfer for 30 s, after which the number of times the worm bent its body to one side was counted for 1 min.
Electrophysiology-Microdissection of C. elegans was performed according to Ref. 27. Membrane currents were recorded in the whole cell configuration using an RK-400 patch-clamp amplifier (Bio-Logic). Acquisition and command voltage were controlled using the pClamp9 software driving a 1322A Digidata (Axon Instruments). Data were analyzed and graphed using Clampfit9 (Axon Instruments) and Origin (OriginLab) software. The resistance of recording pipettes was within 2.5-2.8 megohms. Recordings were performed after 1 min of dialysis only on cells exhibiting resistances above 0.8 megohm. Capacitance and resistance were not compensated. Leak currents were subtracted from all recordings. Currentvoltage relationships were established by measuring the currents at the peak of the current and at the end of the pulse, i.e. 195 ms after the beginning of the pulse. Current-voltage relationships were fitted with Equation 1.
The curves of voltage dependence of the normalized conductance were obtained by dividing I by G max (E Ϫ E rev ) and fitted by a Boltzmann equation.
The bath solution contained 140 mM tetraethylammonium chloride, 6 mM CaCl 2 , 5 mM MgCl 2 , 3 mM 4-aminopyridine, 10 mM HEPES, and sucrose to 333 mOsm (pH 7.2). The pipette solution contained 140 mM CsCl, 5 mM tetraethylammonium chloride, 5 mM HEPES, 4 mM MgATP, 5 mM EGTA, and sucrose to 328 mOsm (pH 7.2). Nemadipine was dissolved in dimethyl sulfoxide at a concentration of 10 mM and then diluted to 1 M in the bath solution. Cells were exposed to nemadipine by placing them at the mouth of a perfusion tube with rapid solution exchange driven by gravity. All chemicals were obtained from Sigma-Aldrich. All experiments were performed at room temperature.

EGL-19
Is the Sole Functional ␣1 Subunit of Voltage-gated Calcium Channels in Body Muscle-By recording in situ currents from body muscle cells of an egl-19 hypomorphic mutant, we previously showed that EGL-19 is the main carrier of voltage-dependent calcium currents (19). However, we could not rule out that other ␣1 subunits are functional in these cells because egl-19-null mutants are lethal, and, at the time, mutants of the other ␣1 subunits were not all available.
Using saline solutions containing potassium channel blockers, we recorded voltage-dependent currents in response to depolarization protocols from muscle cells of wild-type animals and ␣1 subunit mutants. We established the current-voltage relationships at the peak of the current and at the end of the pulse. By fitting the current-voltage relationships with Equation 1 (see "Experimental Procedures"), we obtained for each cell the maximal conductance (G max ), the apparent reversal potential (E rev ), the potential of half-activation (E 0.5 ), and the steepness factor (k) of the current. We first recorded currents from body muscle cells of two ␣1 subunit mutants, CCA-1 and UNC-2. In these mutants, currents were similar to those of the wild type (Fig. 1, A and B). No significant differences were observed for G max , E rev , E 0.5 , and k between wild type and cca-1 or unc-2 mutants (supplemental Table S1).
nca-1 and nca-2 are homologues of the mammalian NALCN which encodes a nonselective cation channel (16). Although selectivity has not been established for NCA-1 or NCA-2 channels, they are likely permeable to calcium. We recorded inward currents from the double mutant nca-2;nca-1, and again we could not detect any differences compared with wild-type currents ( Fig. 1, C and D, and supplemental Table S1).
Taken together, these data indicate that mutations of ␣1 subunits other than EGL-19 do not lead to altered voltage-dependent calcium currents in body muscle cells. These results suggest that EGL-19 is the only functional ␣1 subunit carrying voltage-dependent calcium current in body muscle. Alternatively, compensatory mechanisms (for example, during development) could restore calcium currents in the absence of cca-1, unc-2, nca-1, or nca-2. To rule out this possibility, we acutely blocked calcium currents of wild-type muscles with the drug nemadipine. Nemadipine is a dihydropyridine (DHP), 4 which has been shown to be efficient in nematodes (28). Of the 20 amino acids critical for DHP binding (29), 15 are conserved in EGL-19 whereas five or fewer are conserved in UNC-2, CCA-1, or NCA-1 (Fig. 1E). We therefore assume that the drug is specific to EGL-19. In five cells from wild-type worms, we applied 1 M nemadipine on a depolarizing pulse from Ϫ60 to ϩ20 mV. Currents were inhibited by 99 and 100% at the peak of the current and at the end of the pulse, respectively (Fig. 1F). Altogether, our results strongly suggest that EGL-19 is the sole functional voltage-gated calcium channel ␣1 subunit in body muscle. In the rest of the study, we therefore measured currents only at the peak of amplitude.
UNC-36 Modulates the Voltage Dependence, Activation Kinetics, and Conductance of Calcium Currents in Body Muscle-Which auxiliary subunits regulate EGL-19 calcium currents in body muscle? Two genes, unc-36 and tag-180, encode putative ␣2/␦ subunits in C. elegans (12). Both UNC-36 and TAG-180 contain a von Willebrand factor A domain, conserved in all ␣2/␦ subunits and involved in ␣1 subunit trafficking (30). Strong loss-of-function or null alleles of both ␣2/␦ subunits are available: unc-36(e251) and unc-36(ad698) mutations are nonsense mutations resulting in truncated proteins in the first third of the wild-type protein, whereas tag-180(ok779) is a large deletion skipping four exons and a splice junction (supplemental Fig. S1).
To determine the gross phenotypic effects of ␣2/␦ mutations, we tested locomotion of unc-36 and tag-180 mutants in liquid where animals thrash vigorously. As described previously (31), we found that locomotion is impaired in unc-36 worms. In contrast, tag-180 mutants move similarly to wild type (Fig. 2). To determine the electrophysiological basis for this difference, we recorded voltage-dependent calcium currents in body muscle cells from tag-180 and unc-36 mutants. These currents were unchanged in tag-180 mutants whereas they were altered in unc-36 mutants relative to wild type (Fig. 3, A and B). Fitting the current-voltage relationships in unc-36 mutants revealed a shift of 6 mV toward positive potentials for the half-activation potential (supplemental Table S1), clearly visible when the mean normalized conductance-voltage relationships were plotted (Fig. 3C). In addition, the maximal conductance was decreased by 25% but only in unc-36(ad698) mutants ( Fig. 3B and supplemental Table S1). Finally, activation kinetics of currents were slowed down in unc-36 mutants: time-to-peak measured for the maximal current traces (ϩ20 mV) was slightly increased in unc-36(ad698) and unc-36(e251) compared with the wild type (Fig. 3D). Because of the shift in voltage dependence, we compared time-to-peak values for a range of potentials. At all measured potentials the activation kinetics were slower in unc-36 mutants (supplemental Table S2). We also observed that the progressive decrease of currents during the 200-ms pulse, which may reflect a very slow inactivating process, was significantly faster in unc-36(e251) (Fig. 3D and supplemental Table S2). FIGURE 1. Calcium currents recorded from body muscles of wild-type animals and ␣1 subunit mutants. A, representative traces of inward currents recorded from wild-type, cca-1(ad1650), unc-2(mu74), and unc-2(lj1) worms in response to depolarizing pulses from Ϫ60 to Ϫ20, 0, ϩ20, and ϩ40 mV. B, mean current-voltage relationships Ϯ S.E. (error bars) established for peak (left) and end pulse (right) currents from wild-type, cca-1(ad1650), unc-2(mu74), and unc-2(lj1) worms. Currents were measured in body muscle cells from wild-type (n ϭ 18), unc-2(mu74) (n ϭ 10), unc-2(lj1) (n ϭ 14), and cca-1 (ad1650) (n ϭ 9) animals. C, representative traces of inward currents recorded in wild-type and nca-2(gk5);nca-1(gk9) worms in response to depolarizing pulses from Ϫ60 to Ϫ20, 0, ϩ20, and ϩ40 mV. D, mean current-voltage relationships Ϯ S.E. established for peak (left) and end pulse (right) currents from wild-type and nca-2(gk5);nca-1(gk9) worms. Currents were measured in body muscle cells from wild-type (n ϭ 6) and nca-2(gk5);nca-1(gk9) (n ϭ 5) worms. E, identity of the residues critical for DHP binding in rat ␣1C subunit (NP_036649.2), EGL-19, UNC-2, CCA-1, and NCA-1. The residues critical for DHP binding are distributed in the segment S6 of domains I, III, and IV (IS6, IIIS6, and IVS6), and in the segment S5 of domain III (IIIS5). Of the 20 residues, 15 are conserved in EGL-19, 5 in UNC-2, 5 in CCA-1, and 4 in NCA-1. F, effect of the DHP nemadipine on the inward currents. Currents were obtained from wild-type muscle cells in response to a voltage pulse from Ϫ60 to ϩ20 mV. Nemadipine was added to the extracellular solution at 1 M. unc-36 is expressed in muscle and also in motor neurons (5). The observed changes in unc-36 worms could therefore be an indirect effect due to the absence of the protein in motor neurons and not because of a direct role of UNC-36 in voltage-dependent calcium currents in muscle. To rule out this possibility, we rescued unc-36 specifically either in neurons or in body muscle cells. Whereas the locomotion defect was abolished in worms rescued for unc-36 in neurons (Fig. 2), the half-activation potential was still shifted in these worms (Fig. 3C and supplemental Table S1). In contrast, locomotion was still impaired in worms rescued for unc-36 in muscle (Fig. 2), but the currents were almost identical to those of wild type (Fig. 3C and supplemental Table S1). Activation kinetics of the currents were also altered in worms rescued for unc-36 in neurons, whereas they was similar to those of wild type in worms rescued for unc-36 in muscle ( Fig. 3D and supplemental Table S2). We conclude from these results that UNC-36 is the ␣2/␦ subunit of the voltagegated calcium channel in body muscle cells and that UNC-36 influences the voltage dependence, activation kinetics, and conductance of EGL-19.
CCB-1 Regulates the Conductance and the Voltage Dependence of Calcium Currents in Body Muscle-Two putative ␤ subunits are present in C. elegans, CCB-1 and CCB-2. CCB-1 contains the two conserved ␤ subunit domains necessary for interaction with ␣1 subunit, i.e. the Src homology 3 and guanylate kinase domains (3), whereas CCB-2 does not have the guanylate kinase domain (supplemental Fig. S2). Mutants exhibiting large deletions of these two genes were generated by the International C. elegans Gene Knock-out Consortium: ccb-1(gk18) and ccb-2(ok862). ccb-1(gk18) mutants are 2-fold arrested, and the strain VC37 is therefore maintained as a balanced strain by the translocation szT1. We rescued ccb-1 with a transgene expressing ccb-1 under its own promoter. We verified by PCR that the ccb-1 mutation could be homozygosed and that the balancer was lost in animals carrying the rescue transgene. The rescuing transgene was co-injected with a fluorescent marker (Pccb-1:GFP:H2B) to label cells rescued by the extrachromosomal array. Extrachromosomal arrays are only inherited in a semistable manner; in rescued animals we could therefore identify and record from both ccb-1 rescued cells (GFP-positive cells) and ccb-1 mutant cells (nonfluorescent cells). We selected a viable strain segregating animals with a reasonable number of nonrescued muscle cells.
At the gross level, ccb-2 mutants were phenotypically wild type, whereas ccb-1 rescued worms were smaller and a bit sluggish. Quantitative tests showed that ccb-2 locomotion was similar to wild type whereas locomotion was only partially restored in ccb-1 rescued animals (Fig. 4A).
We analyzed the spatial expression pattern of ccb-2 and ccb-1 by generating promoter-GFP fusions of each promoter. ccb-1 was expressed widely in most neurons and muscle types whereas ccb-2 expression was only observed in a few neurons in the head and in the ventral cord (Fig. 4, B and C).
Consistent with the ccb-2 expression pattern, inward currents recorded from body muscle cells of ccb-2 mutants were not different from wild type (Fig. 5, A-C and G, and supplemental Table S1). In ccb-1 rescued animals we recorded currents from rescued cells (GFP-positive) and nonrescued cells (nonfluorescent). Nonrescued muscle cells exhibit almost no inward current in response to depolarization (Fig. 5D). Analysis of the current-voltage relationship showed that voltage-dependent calcium currents were altered, even in rescued muscle cells (Fig.  5E and supplemental Table S1). Maximal conductance was reduced by 47% in ccb-1 rescued cells relative to wild type. The voltage dependence was strongly altered because the half-activation potential was shifted 5 mV toward positive potentials in ccb-1 rescued cells, and the steepness factor was also altered ( Fig. 5F and supplemental Table S1). Activation time course and inactivation decays were not obviously different in ccb-1 rescued cells (Fig. 5H and supplemental Table S2). Altogether, these results show that CCB-1 is the ␤ subunit of the voltagegated calcium channel EGL-19 in body muscle cells and that CCB-1 plays a crucial role for the proper function of the channel.

DISCUSSION
Taken together, our results show that voltage-dependent calcium channels of C. elegans striated muscles are composed of the ␣1 EGL-19, the ␤ CCB-1, and the ␣2/␦ UNC-36 subunits. Consistent with previous studies (18,19,32,33), our recordings of calcium currents from body muscles display a biphasic appearance, with an early transient peak followed by a slowly inactivating component. At first sight, this could indicate the existence of two types of calcium channels in these cells. Recently, two studies gave opposite results about a putative role of the P/Q-type UNC-2 and the T-type CCA-1 in body muscle: Liu et al. (32) showed that action potentials from cca-1 and unc-2 mutants are altered, whereas Gao and Zhen (33) showed that they are similar to those of wild type. Our study, together with previous work, gives strong evidence that the L-type EGL-19 is the sole functional ␣1 subunit of voltage-gated calcium channels in body muscle. First, inward currents from unc-2 (P/Q-type), cca-1 (T-type) and nca-2;nca-1 mutants are similar to those of the wild type. Second, both the peak and the maintained components of the currents are abolished in the presence of the DHP nemadipine, a drug specific to L-type ␣1 subunit (this study and 32). Third, calcium currents from body muscles measured either at the start or at the end of voltage pulses have both a threshold around Ϫ20 mV and a peak amplitude around ϩ20 mV. In contrast, T-type CCA-1 currents recorded from pharyngeal muscles exhibit a more negative threshold, approximately Ϫ50 mV, and reach a maximal amplitude approximately Ϫ30 mV (15). Fourth, we showed in a previous study that the peak component of the currents is related to a calcium-dependent inactivation process (19). Finally, analysis of expression patterns revealed that unc-2, cca-1, nca-1, and nca-2 are either not expressed in body muscles or restricted to a few anterior-most body muscles (24,27,31). Altogether, these results are in support of EGL-19 being the only functional ␣1 subunit in body muscle cells.
The shift in voltage dependence and the decrease in conductance of calcium currents observed in the ␣2/␦ unc-36 mutants are similar to those reported previously by Fuller-Bicer et al. in mice cardiomyocytes (34). That study was until now the only one describing the effects of ␣2/␦ mutations on L-type currents in native cells from adult animals. However, the effects of ␣2/␦ on calcium current kinetics differ between the two studies: we observe a slight increase in activation kinetics and almost no effect on inactivation whereas they mainly report a strong alteration of inactivation. This discrepancy could naturally be organism-specific (worm versus mouse) but it is also possible that the effects of ␣2/␦ subunits depend on the identity of both ␣2/␦ and L-type ␣1 subunits. For example, depletion of the murine muscle ␣1/␦ by siRNA in muscle cell lines accelerates activation and inactivation kinetics of currents carried by the skeletal ␣1 subunit but slows down inactivation decay of currents carried by the cardiac ␣1 subunit (35,36). The maximal conductance of voltage-dependent calcium currents is decreased only in body muscles from unc-36(ad698) mutants but not from unc- 36(e251). This difference is possibly related to the localization of the two mutations. The e251 mutation is a G-to-A transition, resulting in a stop codon at residue 452 located at the beginning of the Cache domain (25) (supplemental Fig. S1). The von Willebrand factor A domain, which has been shown in mammals to be involved in ␣1 subunit trafficking (30), is retained in this truncated protein, suggesting that the mutated UNC-36 could be partially functional. The ad698 mutation is a C-to-T transition, resulting in a stop codon at residue 412 located in the von Willebrand factor A domain. Such a mutation, contrary to the previous one, could thus impair the function of the von Willebrand factor A domain and alter ␣1 subunit localization. The roles of the different domains of ␣2/␦ are still unclear; thus, the availability of multiple alleles of unc-36 gives an exciting perspective to investigate further the structure/function relationship of ␣2/␦ subunits, first in C. elegans and then, by extrapolation, in other organisms.
Our results from unc-36 mutants are consistent with those obtained by calcium imaging in cultured C. elegans mechanosensory neurons showing that UNC-36 activates EGL-19 (5). A previous study in pharyngeal muscles has, however, reported an inhibitory role of UNC-36 on the calcium influx (21). Whereas the L-type EGL-19 is thought to be the main ␣1 subunit in mechanosensory neurons (5), both EGL-19 and the T-type CCA-1 are known to drive currents in pharyngeal muscles (15). The inhibitory effect of UNC-36 on calcium influx in the pharynx could therefore be a consequence of the interaction between UNC-36 and CCA-1 instead of EGL-19. This hypothesis should be investigated further, especially because the role of ␣2/␦ on native T-type currents is poorly understood and still debated (37)(38)(39). Finally, calcium currents from body muscle of unc-36(e251) mutants have recently been reported to present a voltage dependence similar to that of wild type (33). The difference between our study and their results could be explained by their use of voltage ramp protocols instead of pulses. Generating a current-voltage relationship from voltage ramp protocols can potentially distort the overall shape of the curve (40) and could possibly have masked a slight shift of the voltage dependence in unc-36 mutants.
In mice and zebrafish, knock-out animals of ␤ subunits of L-type voltage-gated calcium channels die before reaching adulthood (4,41,42). In C. elegans, ccb-1-null mutants are embryonic lethal, but we took advantage of mosaic rescue to study, for the first time in adult animals, the effects of the lack of ␤ subunit in striated muscle cells. No voltage-dependent calcium currents were recorded in nonrescued muscle cells, suggesting that without the ␤ subunit the ␣1 subunit EGL-19 is not functional at all. In vertebrate fetal cardiac or skeletal muscle cells, residual voltage-dependent calcium currents were observed in the absence of the main ␤ subunit expressed in these tissues (42,43). However, compensatory mechanisms could not be ruled out because three of four genes encoding ␤ subunits in mammals are expressed in cardiac and skeletal muscle cells (3). In our case, redundancy is highly unlikely because (i) the only other gene encoding ␤ subunit in C. elegans, ccb-2, is not expressed in muscle and (ii) the CCB-2 protein lacks the guanylate kinase domain essential for ␣1/␤ interactions (3).
Our recordings from ccb-1 rescued cells show a decrease in amplitude and a shift in voltage dependence, similar to those observed in fetal vertebrate ␤ knock-out cells (42,43) or in various expression systems (3). However, it is difficult to make detailed interpretations of the currents in rescued ccb-1 cells because the expression level and regulation of the rescuing CCB-1 transgene from an extrachromosomal array are likely very different from the endogenous channel subunit. This could explain why the rescued animals are not fully wild type. Either low expression or overexpression of ccb-1 could induce changes in voltage-dependent calcium currents. In the case of a lower expression of ccb-1, because ␣1/␤ interaction is reversible (44), there could be enough CCB-1 to transport the nascent EGL-19 to the plasma membrane but not enough to keep all EGL-19 associated with CCB-1. It is, however, more likely that FIGURE 5. Calcium currents recorded from body muscles of wild-type animals and ␤ subunit mutants. A, representative traces of inward currents recorded in wild-type and ccb-2(ok862) worms in response to depolarizing pulses from Ϫ60 to Ϫ20, 0, ϩ20, and ϩ40 mV. B, mean current-voltage relationships Ϯ S.E. (error bars) of currents from wild type and ccb-2 mutants. Currents were measured in body muscle cells from wild-type (n ϭ 9) and ccb-2(ok862) (n ϭ 11) worms. C, voltage dependence of the normalized conductance in wild type and ccb-2 mutants. The mean normalized conductance Ϯ S.E. was plotted as a function of voltage. D, representative traces of inward currents from body muscles of wild-type and ccb-1 rescued animals in response to depolarizing pulses from Ϫ60 to Ϫ20, 0, ϩ20, and ϩ40 mV. In ccb-1 rescued animals, currents were recorded from rescued muscle cells (cell ϩ) expressing the Pccb-1:ccb-1 construct or from nonrescued cells (cell Ϫ). E, mean current-voltage relationships Ϯ S.E. of currents from wild-type and ccb-1 rescued animals. Currents were measured in body muscle cells of wild type (n ϭ 10), positive cells from ccb-1 rescued (n ϭ 8), and negative cells from ccb-1 rescued animals (n ϭ 4). F, voltage dependence of the normalized conductance in wild-type and ccb-1 rescued animals. The mean normalized conductance Ϯ S.E. was plotted as a function of voltage from muscle cells of wild-type and positive cells from ccb-1 rescued animals. G, kinetics of inward currents of wild type and ccb-2 mutants. Time-to-peak (left) and fractional inactivation at the end of the pulse (R 200 , right) were measured for the maximal current traces (ϩ20 mV). R 200 represents the inactivated fraction of current (1 Ϫ I 200ms /I peak ). Mean values Ϯ S.E. are plotted. Both were similar in wild type and ccb-2 mutants (Mann-Whitney test, p ϭ 0.055 and p ϭ 0.91, respectively). H, kinetics of inward currents of wild-type and ccb-1 rescued animals. Time-to-peak (left) and fractional inactivation at the end of the pulse (R 200 , right) were measured for the maximal current traces (ϩ20 mV). R 200 represents the inactivated fraction of current (1 Ϫ I 200ms /I peak ). Mean values Ϯ S.E. are plotted. Both were similar in wild-type and ccb-1 rescued animals (Mann-Whitney test, p ϭ 0.15 and p ϭ 0.97, respectively). ccb-1 is in fact overexpressed in ccb-1 rescued cells because the transgene is a multicopy array. In this case, excess CCB-1 could be available for channel-independent functions: ␤ subunits have been shown in mammals to modulate gene expression by interacting with transcription factors or heterochromatin-associated proteins (45)(46)(47). Although purely speculative, it is, for example, possible that the CCB-1 subunit could recruit posttranscriptional modification machinery (for example, a kinase that phosphorylates the channel), resulting in changes to channel properties. Regardless of the mechanism, viable ␤ knockout animals with no redundancy represent exciting tools for future studies into how voltage-gated calcium currents are regulated.