gamma 1 Subunit Interactions within the Skeletal Muscle L-type Voltage-gated Calcium Channels*

Jyothi ArikkathDagger , Chien-Chang ChenDagger , Christopher Ahern§, Valérie AllamandDagger , Jason D. FlanaganDagger , Roberto Coronado§, Ronald G. Gregg, and Kevin P. CampbellDagger ||

From the Dagger  Howard Hughes Medical Institute, Departments of Physiology and Biophysics and Neurology, University of Iowa, Iowa City, Iowa 52242, the § Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, and the  Departments of Biochemistry and Molecular Biology and Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky 40202

Received for publication, August 23, 2002, and in revised form, October 28, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Voltage-gated calcium channels mediate excitationcontraction coupling in the skeletal muscle. Their molecular composition, similar to neuronal channels, includes the pore-forming alpha 1 and auxiliary alpha 2delta , beta , and gamma  subunits. The gamma  subunits are the least characterized, and their subunit interactions are unclear. The physiological importance of the neuronal gamma  is emphasized by epileptic stargazer mice that lack gamma 2. In this study, we examined the molecular basis of interaction between skeletal gamma 1 and the calcium channel. Our data show that the alpha 11.1, beta 1a, and alpha 2delta subunits are still associated in gamma 1 null mice. Reexpression of gamma 1 and gamma 2 showed that gamma 1, but not gamma 2, incorporates into gamma 1 null channels. By using chimeric constructs, we demonstrate that the first half of the gamma 1 subunit, including the first two transmembrane domains, is important for subunit interaction. Interestingly, this chimera also restores calcium conductance in gamma 1 null myotubes, indicating that the domain mediates both subunit interaction and current modulation. To determine the subunit of the channel that interacts with gamma 1, we examined the channel in muscular dysgenesis mice. Cosedimentation experiments showed that gamma 1 and alpha 2delta are not associated. Moreover, alpha 11.1 and gamma 1 subunits form a complex in transiently transfected cells, indicating direct interaction between the gamma 1 and alpha 11.1 subunits. Our data demonstrate that the first half of gamma 1 subunit is required for association with the channel through alpha 11.1. Because subunit interactions are conserved, these studies have broad implications for gamma  heterogeneity, function and subunit association with voltage-gated calcium channels.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The L-type voltage-gated calcium channels of the skeletal muscle serve both as a voltage-gated calcium channel and as a voltage sensor for excitation-contraction (EC)1 coupling (1, 2). These channels serve to couple depolarization to intracellular calcium release via the ryanodine receptor. The sites of EC coupling in the skeletal muscle are the triads, which are highly organized junctions comprising the t-tubules and the underlying sarcoplasmic reticulum (3). The voltage-gated calcium channels are localized predominantly in the t-tubules in close association with the ryanodine receptor in the sarcoplasmic reticulum (4, 5).

At the molecular level, the calcium channel is composed of the pore-forming alpha 11.1 subunit and auxiliary alpha 2delta , beta 1, and gamma 1 subunits (6). This four-subunit composition of the channels is similar to that of the neuronal voltage-gated channels (7, 8). The auxiliary alpha 2delta and beta  subunits enhance membrane trafficking of the alpha 11.1 subunit and modulate the voltage-dependent kinetics of the channel (9). In addition to the role of the beta 1 subunit in the trafficking of the channel, it also has a crucial role in EC coupling as emphasized by the absence of EC coupling and early lethality in mice that lack the skeletal beta 1 subunit (10). The subunit interactions of the alpha 2delta and beta  subunits have been relatively well defined.

The gamma 1 subunit is an auxiliary subunit with four predicted transmembrane domains and intracellular N and C termini. The role of the gamma  subunit in the membrane trafficking of the channels and subunit interaction remain unclear. The gamma 1 subunit was the only gamma  subunit that was originally known and is associated with the skeletal muscle voltage-gated calcium channel (11, 12). However, the identification of a neuronal gamma 2 subunit (13) renewed interest in the gamma  subunits and led to the identification of a number of gamma  subunits (14-16).

The beta  subunits of the voltage-gated calcium channels are known to be capable of forming heterogenous complexes both in vivo (17, 18) and in vitro. The beta  subunits and the alpha 1 subunit possess highly conserved interaction regions (19) allowing the formation of heterogeneous channel complexes. The alpha 2delta subunits modulate different alpha 1 subunits in vitro (20), and presumably this heterogeneity extends to the in vivo situation. Such diversity of interaction would potentially multiply the number of possible combinations of channels with different biophysical and physiological properties, which could then translate into fine modulation of a variety of cellular responses.

gamma subunits are relatively less well understood. Subunit interactions of gamma  subunits with the voltage-gated calcium channels are unknown, and their ability to form heterogeneous complexes is unclear. To address these questions, we took advantage of gamma 1 null mice. Mice that lack gamma 1 have been generated by use of conventional gene targeting strategy (21, 22). The mice have no detectable phenotype, and we have demonstrated previously (21) that the subunits of the skeletal muscle calcium channel are expressed at wild type levels in the gamma 1 null mice. The lack of the gamma 1 subunit also does not appear to diminish the ability of the other subunits to assemble together and conduct voltage-gated calcium currents. However, the absence of the gamma 1 subunit slows the inactivation kinetics and increases the amplitude (21, 22) of L-type currents in the skeletal muscle. This led us to examine if the L-type calcium channel is maintained as a complex in the gamma 1 null mice. We hypothesized that if the channel were still maintained as a complex in the gamma 1 null mice, it would serve as a valuable tool offering the potential to explore subunit-subunit interactions in an in vivo environment.

Our data show that the skeletal L-type channel is indeed maintained as a complex in the gamma 1 null mice. To examine the ability of the gamma  subunits to form heterogeneous complexes, the gamma 1 and gamma 2 subunits were introduced into the skeletal muscle of the gamma 1 null mice via adenovirus-mediated expression, and their ability to incorporate into the channel was examined. We demonstrate that the gamma 1 does incorporate, but the gamma 2 does not. Consistent with these results, gamma 1EGFP localizes to the t-tubules, whereas the gamma 2EGFP does not demonstrate organized t-tubule localization. Furthermore, by using a gamma 1/gamma 2 chimeric strategy, we demonstrate that the first half of the gamma 1 subunit mediates its interaction with the calcium channel. Examination of the electrophysiological characteristics of the chimeric subunits in gamma 1 null myotubes showed that the first half of the protein is sufficient to restore calcium conductance of L-type currents in gamma 1 null myotubes.

To examine the subunit of the channel with which the gamma 1 subunit associates, we examined the channel in the muscular dysgenesis (mdg) mice. In the absence of alpha 11.1 in the mdg mice, gamma 1 does not associate with alpha 2delta , indicating that alpha 11.1 is necessary for gamma 1 to associate with the calcium channel. These predictions are confirmed by coimmunoprecipitation of alpha 11.1 and gamma 1EGFP subunits from transiently transfected cells.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Enrichment and Purification of the Skeletal Voltage-gated Calcium Channels-- KCl microsomes were prepared from wild type and gamma 1 null mice as described previously (23). The microsomes were solubilized twice in a buffer containing 50 mM Tris, 1% digitonin, 0.5 M NaCl, and seven protease inhibitors (benzamidine, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, calpain inhibitor, calpeptin, and pepstatin). The solubilized material was subjected to wheat germ agglutinin (WGA) chromatography. The material bound to the WGA column was eluted using N-acetylglucosamine and concentrated. In some cases, the material was used as the enriched voltage-gated calcium channel. For purification, the WGA-eluted material was diluted and applied to a diethylaminoethylcellulose column, and the bound material eluted with buffer containing 50 mM Tris, 0.1% digitonin, and 100 mM NaCl.

Constructs and Generation of Adenoviruses-- All the adenoviruses and cell expression constructs used in this study expressed the protein under the control of the cytomegalovirus promoter. The cDNAs of interest were subcloned into the pAd5CMVK-NpA vector and used for generation of the adenovirus by standard homologous recombination techniques. The recombinant viruses were then purified using standard techniques. The Gene Transfer Vector Core at the University of Iowa, Iowa City, IA, generated all adenoviruses in this study. In some cases, the cDNAs in the adenovirus vectors were used for cell expression studies.

gamma 1-- The rabbit gamma 1 subunit (GenBankTM accession number M32231) was subcloned into the BamHI/BamHI site of the adenovirus shuttle vector. The resulting construct was checked for appropriate orientation by restriction analysis.

gamma 2-- The mouse gamma 2 subunit (GenBankTM accession number NM_007583) was excised from the pCDNA3 vector using HindIII and BamHI and subcloned into the HindIII/BamHI sites of the adenovirus vector.

gamma 1EGFP and gamma 2EGFP-- The gamma 1EGFP and gamma 2EGFP constructs were generated by inserting the cDNA into the pEGFP-N1 vector (Clontech) in-frame with the enhanced fluorescent green protein (EGFP) using standard PCR techniques. The resulting construct encoded the gamma  subunit with the EGFP tag at the C terminus of the protein. The resulting constructs were excised and subcloned into the adenovirus vector.

gamma 1/gamma 2 Chimera-- To generate this construct, a fragment of the rabbit gamma 1 cDNA was amplified using a forward primer that includes a HindIII site and the start codon (gamma 1 forward, 5' CCC AAG CTT CCA CCA TGT CCC CGA CGG AAG CC 3') and the reverse primer in the region after the second transmembrane domain (5' GTT GTG GCG TGT CTT CCG CTT CTT CCT GAA GGC 3'). This reverse primer also includes a stretch of residues homologous to the gamma 2 subunit. Another PCR fragment was generated using the gamma 2 cDNA as a template, with the forward primer (5' GCC TTC AGG AAG AAG CGG AAG ACA ACG CCA CAA C 3') and a reverse primer that includes a stop codon and a BamHI site (gamma 2 reverse, 5' CGG GAT CCC GTC ATA CGG GCG TGG TCC GGC G 3'). The products from the two PCRs were mixed and reamplified using the gamma 1 forward and gamma 2 reverse primers. The resulting product was subcloned into the adenovirus shuttle vector into the HindIII and BamHI sites. The final construct was sequenced to verify integrity. This construct encodes a protein that includes the N terminus to aa 133 of gamma 1 and aa 129 through the C terminus of the gamma 2 subunit. This protein is detected by an antibody to the gamma 2 subunit.

gamma 2/gamma 1 Chimera-- A fragment of the gamma 2 cDNA was amplified using the forward primer that includes a HindIII site and the start codon (gamma 2 forward, 5' CCC AAG CTT CCA CCA TGG GGC TGT TTG ATC GAG 3') and the reverse primer (5' CGG CCG CAG CAG GTA ATC GTA GAA CTC GCT CGC CGC 3'). A fragment of the gamma 1 cDNA was amplified using the forward primer (5' GCG AGC GAG TTC TAC GAT TAC CTG CTG CGG CCG 3') and a reverse primer that includes a stop codon and a BamHI site (gamma 1 reverse, 5' CGT GGA TCC CGT TAA TGC TCG GGT TCG GC 3'). The products from the two PCRs were mixed and reamplified with the gamma 2 forward and gamma 1 reverse primers. The resulting product was subcloned into the adenovirus shuttle vector into the HindIII and BamHI sites. The final construct was sequenced to verify the integrity. This construct encodes a protein that includes the N terminus to aa 128 followed by aa 134 through the C terminus of the gamma 1 subunit. This protein is detected by an antibody to the gamma 1 subunit.

gamma 1sspnEGFP Chimera-- This construct was generated by standard PCR techniques to replace the first extracellular loop of the gamma 1EGFP with the first extracellular loop of the unrelated four-transmembrane protein sarcospan (24). The first extracellular loop of sarcospan includes the six amino acids RTDPFW. The protein can be detected by an antibody to GFP.

alpha 11.1-- This construct has been described previously (25).

Injection of Adenoviruses into Skeletal Muscle of the gamma 1 Null Mice-- 10 µl of primary particles or diluted secondary particles (~1011 particles/ml) of the adenovirus were injected into the tibialis anterior and the quadriceps muscles of 2-5-day-old gamma 1 null pups as described previously (26). 3 to 5 weeks later, the mice were sacrificed and the injected muscles collected. The tissue was processed for immunohistochemistry or biochemical analysis as described below.

Sucrose Gradient Fractionation-- Wheat germ agglutinin-enriched material or KCl microsomes that were solubilized as described in the methods for the purification of the channel complex were concentrated and loaded on 5-30% linear sucrose gradients with 0.1% digitonin and 0.5 M NaCl. The gradients were centrifuged for 90 min at 50,000 rpm in a 65.2 Ti rotor (Beckman Instruments). 800-µl fractions were collected from the top from each tube.

SDS-PAGE and Immunoblotting-- Samples were subjected to SDS-PAGE on gradient gels and transferred to polyvinylidene difluoride membranes for immunoblotting. The membranes were blocked and incubated with the primary antibody, followed by secondary antibody and detected by enhanced chemiluminescence.

Antibodies-- The antibodies used in this study have been described previously, IID5E1, IIF7 (alpha 11.1 (26)), sheep 6 (alpha 11.1 and beta 1a (27)), guinea pig 1 (alpha 2delta ), guinea pig 11/15/16/77 (gamma 1 (28)), rabbit 239 (gamma 2/gamma 3 (13)), and AP83 (beta -dystroglycan (29)).

Immunofluorescence Analysis-- Tissue samples (quadriceps) were frozen and processed for immunohistochemistry as described previously (26). The sections were labeled with a rabbit polyclonal antibody to beta -dystroglycan (AP83). To visualize the alpha 11.1 subunit, the sections were labeled with the monoclonal antibody to the alpha 11.1 subunit (IIID5E1). To minimize the background, the sections were first treated with the Fab fragment using the conditions described by the manufacturer (The Jackson Laboratories). Cy3-conjugated secondary antibodies were used to detect the primary antibodies. EGFP fluorescence was used to detect the gamma  subunits. Immunolabeled sections were visualized with the ×60 objective using confocal microscopy (Bio-Rad). The bar represents 10 µm.

Primary Cultures and cDNA Transfection-- Primary cultures were prepared from enzyme-digested hind limbs of late-gestation (E18) gamma 1 null embryos. Myoblasts were isolated by enzymatic digestion with 0.125% (w/v) trypsin and 0.05% (w/v) pancreatin. After centrifugation, mononucleated cells were resuspended in plating medium containing 78% Dulbecco's modified Eagle's medium with low glucose, 10% horse serum, 10% fetal bovine serum, and 2% chick embryo extract. Cells were then plated at a density of ~1 × 104 cells per dish on gelatin-coated plastic culture dishes (BD Biosciences). Cultures were grown at 37 °C in 8% CO2, and after the fusion of myoblasts (5-7 days), the medium was replaced with fetal bovine serum-free medium, and CO2 was decreased to 5%. In the case of cDNA expression, gamma  subunit cDNAs of interest and a separate plasmid encoding the T-cell membrane antigen CD8 were mixed at a 1:1 weight ratio and cotransfected with the polyamine LT-1 (Panvera, WI). CD8-transfected cells were identified by incubation with anti-CD8 antibody beads (Dynal, Norway).

Whole-cell Voltage Clamp and Solutions-- Cells were voltage-clamped ~48 h after transfection. Transfected cells revealed by CD8 beads were voltage-clamped with an Axopatch 200B amplifier (Axon Instruments, CA) and a Digidata 1200 (Axon) pulse generator and digitizer. Linear capacitance, leak currents, and effective series resistance were compensated with the amplifier circuit. The voltage dependence of the Ca2+ conductance was fitted according to a Boltzmann distribution, A = Amax/(1 + exp(-(V - V1/2)/k)). Where Amax is Gmax; V1/2 is the potential at which A = Amax/2; and k is the slope factor. The external solution in all cases was (in mM) 130 triethanolamine methanesulfonate, 10 CaCl2, 1 MgCl2, 0.001 tetrodotoxin (Sigma), and 10 HEPES titrated with triethanolamine(OH) to pH 7.4. The pipette solution was (in mM) 140 Cs+-aspartate, 5 MgCl2, 5 EGTA, 10 MOPS-CsOH, pH 7.2.

Analysis of mdg and Wild Type Muscle-- Skeletal muscle from mdg pups or 1- to 2-day-old wild type mice were obtained, and KCl-washed microsomes were prepared as described above. The skeletal muscle calcium channel was enriched using a wheat germ agglutinin column after solubilization as described above. The enriched material was subjected to sucrose gradient fractionation as described, and the fractions were analyzed by SDS-PAGE and immunoblotting.

Transient Transfection in tsA201 Cells and Immunoprecipitation-- tsA201 cells were transfected using the calcium phosphate method. Cells were lysed in buffer containing 50 mM Tris, 1% digitonin, 0.5 M NaCl, and protease inhibitors, and the solubilized material was isolated by centrifugation. The solubilized material was immunoprecipitated with beads to which the antibody to the alpha 11.1 subunit (IIF7) was coupled. The beads were then thoroughly washed in a buffer containing 0.1% digitonin, 0.5 M NaCl, 50 mM Tris, and two protease inhibitors (benzamidine and phenylmethylsulfonyl fluoride). The bound material was eluted using SDS loading buffer and immunoblotted. Aliquots of the cell lysate were also immunoblotted to examine the expression of the proteins.

Tunicamycin Treatment-- TsA201 cells were transfected as described above. Tunicamycin (10 µg/ml) was then added to the medium, the cells were incubated for 2 days and immunoprecipitated as described above.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Skeletal Muscle L-type Calcium Channel Complex Is Maintained in gamma 1 Null Mice-- The generation of gamma 1 null mice using conventional gene targeting strategy has been described previously (21). The subunits of the calcium channel are expressed at normal levels in these mice. This, in conjunction with the expression of robust voltage-gated calcium currents in the skeletal muscle (21, 22), led us to examine if the calcium channel subunits are maintained as a complex. The calcium channel was purified from the wild type and gamma 1 null mice as described under "Materials and Methods." The presence of all the subunits in the complex was verified by Western blotting with antibodies specific to each subunit of the channel (Fig. 1). The gamma 1 subunit can be detected in the complex from the wild type mice but not in the gamma 1 null mice. The alpha 11.1, alpha 2delta , and beta 1a subunits can be detected in the purified material from both the wild type and the gamma 1 null mice, demonstrating that the residual calcium channel is maintained as a complex in the gamma 1 null mice.


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Fig. 1.   The skeletal muscle L-type calcium channel is maintained as a complex in the gamma 1 null mice. Purified calcium channels from the wild type (WT) and gamma 1 null mice were immunoblotted to detect the presence of the subunits. The alpha 11.1, alpha 2, and beta 1a subunits are detected in both the wild type and the gamma 1 null mice, whereas gamma 1 is only detected in the wild type mice, indicating that the channel is maintained as a complex in gamma 1 null mice.

gamma 1 Can Be Stably Incorporated into the L-type Calcium Channels of gamma 1 Null Mice-- The gamma 1 subunit was reintroduced into the skeletal muscle of the gamma 1 null mice, via recombinant adenoviruses, to determine whether it could be incorporated into the calcium channel of the gamma 1 null mice. Expression of the protein was examined by immunoblot analysis (Fig. 2A). The muscle from the wild type mice showed expression of the gamma 1 protein, whereas the gamma 1 null mice did not express any gamma 1 protein. Robust expression of gamma 1 recombinant protein was detected in the muscle of the mice injected with the recombinant adenovirus, confirming that the adenovirus-mediated expression may be successfully used for the expression of this subunit in the skeletal muscle.


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Fig. 2.   Incorporation of an adenovirally expressed gamma 1 into skeletal L-type calcium channel of gamma 1 null mice. A, adenovirally expressed gamma 1 in gamma 1 null skeletal muscle was detected by immunoblot analysis. The blot was reprobed with an antibody to alpha 2. WT, wild type. B, calcium channels from solubilized microsomes were enriched using WGA chromatography and immunoblotted. The channel is enriched by WGA as indicated by the presence of alpha 2 in the WGA elution. gamma 1 is also enriched by the WGA, suggesting association with the calcium channel. C, WGA-enriched material was subjected to linear sucrose gradient fractionation and immunoblot analysis. alpha 11.1, alpha 2, and beta 1a subunits comigrate on the sucrose gradient. gamma 1 migrates in the same fractions, confirming incorporation into the calcium channels of the gamma 1 null mice. D, muscle sections from gamma 1EGFP injected muscle were double labeled with an antibody to the sarcolemma protein, beta -dystroglycan (beta  DG). E, muscle sections were double-labeled with an antibody to alpha 11.1. gamma 1EGFP is localized to the t-tubules and colocalizes with alpha 11.1.

To examine if the virally expressed gamma 1 is incorporated into the residual calcium channel complex of gamma 1 null mice, a WGA-enriched preparation containing highly enriched material was examined for the presence of the gamma 1. gamma 1 is enriched by the WGA column, as is the rest of the channel as indicted by the presence of the alpha 2delta subunit in the elution. This suggests that gamma 1 may be incorporated into the channel complex (Fig. 2B). To confirm this further, the enriched material was subjected to sucrose gradient fractionation and immunoblot analysis (Fig. 2C). Our results clearly demonstrate that gamma 1 cofractionates with the other subunits of the calcium channel, indicating that indeed it is stably incorporated into the calcium channel complex of gamma 1 null mice. A fraction of gamma 1 is not associated with the channel. It is possible that this pool represents free gamma 1 that is not complexed with the channel due to overexpression of the protein, because it is not observed in similar preparations from older wild type skeletal muscle (data not shown).

To examine the localization of gamma 1, a construct that encodes gamma 1 with an EGFP at the C terminus was engineered. Expression of this protein in mammalian cells revealed that gamma 1EGFP traffics to the plasma membrane, even in the absence of the other subunits of the calcium channel (data not shown). By using adenovirus-mediated expression, the EGFP tagged constructs were introduced in the muscle of the gamma 1 null mice. Interestingly, the protein is also localized to the sarcolemma (Fig. 2D), as indicated by its colocalization with beta -dystroglycan, a marker for the sarcolemma (30) (Fig. 2D). In addition, gamma 1EGFP is localized to the t-tubules (Fig. 2, D and E), as confirmed by its colocalization with alpha 11.1 (Fig. 2E).

gamma 2 Does Not Incorporate into the Skeletal L-type Calcium Channels of gamma 1 Null Mice-- The ability of gamma 2 to incorporate into the calcium channel of gamma 1 null mice was then examined using a similar approach. The adenovirus encoding gamma 2 allows robust expression of the protein in the skeletal muscle of gamma 1 null mice. There is no detectable endogenous protein in the skeletal muscle of either wild type or gamma 1 null mice (Fig. 3A) as reported previously (13).


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Fig. 3.   gamma 2 subunit is not incorporated into the skeletal L-type calcium channel of gamma 1 null mice. A, adenovirally expressed gamma 2 in gamma 1 null skeletal muscle was detected by immunoblot analysis. The blot was reprobed with an antibody to alpha 2. WT, wild type. B, microsomes from the gamma 1 null mice expressing gamma 2 were solubilized, and the channel was enriched using WGA chromatography. The calcium channel is enriched by the WGA as indicated by the presence of the alpha 2 subunit in the WGA elution. The gamma 2 subunit is not enriched by the WGA. C, solubilized microsomes were subjected to linear sucrose gradient fractionation and immunoblot analysis. alpha 11.1, alpha 2, and beta 1a subunits comigrate on the sucrose gradient. gamma 2 subunit does not comigrate with the calcium channel subunits, confirming that it is not incorporated into the channel of gamma 1 null mice. D, muscle sections from gamma 2EGFP-injected muscle were double-labeled with an antibody to the sarcolemma protein, beta -dystroglycan (beta  DG). gamma 2EGFP localizes to the sarcolemma. E, muscle sections were double-labeled with an antibody to alpha 11.1. gamma 2EGFP is not localized to the t-tubules and shows a punctate pattern. gamma 2EGFP does not colocalize with alpha 11.1.

WGA enrichment of the channel complex indicates that, unlike gamma 1, gamma 2 is in the void, indicating that that gamma 2 may not be incorporated into the complex (Fig. 3B). To ascertain if the gamma 2 subunit is weakly associated with the complex and the steps leading to the WGA chromatography disrupt this association, sucrose gradient fractionation of solubilized microsomes from gamma 2 injected mice was examined for the presence of gamma 2 and the other subunits of the channel (Fig. 3C). These results demonstrate that gamma 2 does not comigrate in the same fractions as the rest of the channel complex confirming that it is not incorporated into the channel complex in gamma 1 null mice.

Similar to gamma 1EGFP, gamma 2EGFP traffics to the plasma membrane in transiently transfected mammalian cells (data not shown). Consistent with the biochemical data indicating the absence of an association of gamma 2 with the calcium channel, the adenovirally expressed gamma 2EGFP in the skeletal muscle of the gamma 1 null mice does not demonstrate an organized t-tubule expression pattern (Fig. 3D). Interestingly, similar to that observed with the gamma 1 subunit, the protein is also localized to the sarcolemma (Fig. 3D) and colocalizes with beta -dystroglycan, a marker for the sarcolemma (Fig. 3E). In contrast to gamma 1, gamma 2 appears in punctate clusters within some muscle fibers (Fig. 3E). In other fibers, a more diffused pattern is observed (data not shown). However, the protein is not localized to the t-tubules as confirmed by the absence of colocalization with the alpha 11.1 subunit (Fig. 3E).

Chimeras of gamma 1 and gamma 2 Define Interaction Domain of gamma 1 with the Calcium Channel-- The gamma  subunits possess a similar four-transmembrane domain structure with intracellular N and C termini. The first extracellular loops of the gamma  subunits have charged regions that are conserved across the gamma  subunits (12). We therefore hypothesized that the first half of the gamma 1 subunit, including the first two transmembrane domains, might mediate the interaction of the protein with the channel, placing the first loop in close proximity to the channel.

To test this hypothesis, we took advantage of the ability of the gamma 1 subunit, but not the gamma 2 subunit, to incorporate into the calcium channel. Chimeric proteins containing the first two transmembrane domains of the gamma 1 subunit and the last two domains of the gamma 2 subunit were generated (Fig. 4A). The ability of this protein to incorporate into the channel was examined by techniques similar to those described for the gamma 1 and gamma 2 subunits. The chimeric protein can be detected in microsomes from the injected muscle (Fig. 4B).


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Fig. 4.   The first half of the gamma 1 subunit interacts with the L-type calcium channels. A, schematic of the gamma 1/gamma 2 chimera. The protein encodes the first half of the gamma 1 subunit, including the N terminus, first extracellular loop, and first two transmembrane domains, fused to the second half of the gamma 2 subunit. B, adenovirally expressed gamma 1/gamma 2 in gamma 1 null skeletal muscle was detected by immunoblot analysis. The blot was reprobed with an antibody to the alpha 2 subunit. WT, wild type. C, solubilized microsomes were subjected to sucrose gradient fractionation and immunoblot analysis for subunits of the calcium channel. The gamma 1/gamma 2 comigrates with the subunits of the calcium channel, indicating that it is incorporated. D, schematic of the gamma 2/gamma 1 chimera. The protein encoded includes the first half of the gamma 2 subunit followed by the second half of the gamma 1 subunit. E, adenovirally expressed gamma 2/gamma 1 in gamma 1 null skeletal muscle was detected by immunoblot analysis. The blot was reprobed with an antibody to the alpha 2 subunit. F, microsomes from gamma 1 null mice expressing gamma 2/gamma 1 were solubilized, and the channels were enriched using WGA. The gamma 2/gamma 1 chimera is in the void of the WGA, whereas the channel is in the WGA elution as indicated by the presence of the alpha 2, indicating that the gamma 2/gamma 1 chimera is not incorporated.

To examine incorporation of the chimeric gamma 1/gamma 2 protein into the calcium channel, the microsomes from such a preparation were solubilized and subjected to sucrose gradient fractionation and immunoblotting. Our data show that the gamma 1/gamma 2 chimera comigrates in the same fractions as the other subunits of the channel, confirming that it is indeed incorporated into the channel complex (Fig. 4C). Similar to that observed for the gamma 1 studies described above, in some experiments a pool of the gamma 1/gamma 2 chimera that is not associated with the channel complex is observed (data not shown), presumably as a result of overexpression.

To assess if secondary interaction sites exist on the gamma 1 subunit that allow interaction with the calcium channel, we generated a construct that encodes the first two transmembrane domains of the gamma 2 subunit and the last two transmembrane domains of the gamma 1 (Fig. 4D). Similar to the other adenovirally expressed proteins, the gamma 2/gamma 1 chimera showed robust expression in the skeletal muscle of the gamma 1 null mice (Fig. 4E). The gamma 2/gamma 1 protein is not enriched with the calcium channel as indicated by its presence in the void of the WGA column, whereas the other subunits of the calcium channel are in the WGA elution, as indicated by the presence of the alpha 2delta subunit (Fig. 4F). These results clearly demonstrate that the gamma 2/gamma 1 chimeric protein does not incorporate into the calcium channel of the gamma 1 null mice.

gamma 1 and gamma 1/gamma 2 Chimera Restore Conductance of L-type Calcium Channels in gamma 1 Null Myotubes-- We have demonstrated previously (21) that the L-type voltage-gated calcium current conductance is increased in gamma 1 null myotubes. To examine the functional effects of the gamma  subunits and the chimeras, the different constructs were introduced into the myotubes of the gamma 1 null mice (Fig. 5, A and B). In agreement with the biochemical studies, re-expression of gamma 1 and gamma 1/gamma 2 chimera reduced the calcium conductance (142 ± 12.1 and 160.5 ± 16.7 pS/pF, respectively) significantly compared with that of gamma 1 null myotubes (296.1 ± 10.8 pS/pF). Consistent with the lack of incorporation of gamma 2 and gamma 2/gamma 1, these chimeras did not alter the conductance significantly (262.6 ± 20.4 and 276.4 ± 8.2 pS/pF, respectively). In all of the cases, no effect was observed on the voltage dependence of current conductance (G-V curve), the voltage of half-maximal conductance, V1/2, and slope factor, k values (Table I). These results demonstrate that the first half of gamma 1, which mediates subunit interaction, can modulate the functional properties of skeletal L-type voltage-gated calcium channels.


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Fig. 5.   Electrophysiological characterization of the gamma  subunits in gamma 1 null myotubes. A, whole-cell calcium currents when gamma 1 null or gamma 1 null myotubes transfected with the indicated constructs are depolarized to 0, +20, or +40 mV from a holding potential of -40 mV. The pulse duration was 500 ms. B, voltage dependence of calcium conductance for gamma 1 null myotubes or gamma 1 null myotubes transfected with the indicated constructs. The curves correspond to a Boltzmann fit of the population mean with the parameters Gmax (pS/pF), V1/2 (mV), and k (mV), respectively, of 301.7, 19.8, and 6.1 for gamma 1 null; 142.2, 21.4, and 5.8 for gamma 1; 263.3, 16.1, and 5.9 for gamma 2; 153.6, 20.5, and 4.8 for gamma 1/gamma 2 chimera; and 274.9, 18.2, and 5.9 for gamma 2/gamma 1.

                              
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Table I
Ca2+ conductance of the gamma 1 null cells expressing gamma 1, gamma 2, gamma 1/gamma 2, and gamma 2/gamma 1 subunits
Entries correspond to mean ± S.E. of Boltzmann parameters fitted to each cell. The number of cells is in parentheses.

gamma 1 Directly Interacts with alpha 11.1-- To determine the subunit of the calcium channel that interacts with the gamma 1 subunit, we took advantage of the muscular dysgenesis (mdg) mice. The mdg mouse arose as a spontaneous mutation in the gene that encodes the alpha 11.1 (31) that results in the loss of any detectable protein (32). In the absence of alpha 11.1, alpha 2delta (32, 33) and beta 1a are still expressed. We sought to examine gamma 1 subunit in these mice and determine whether, in the absence of alpha 11.1, gamma 1 is associated with alpha 2delta . The WGA-enriched material from mdg and 1-2-day-old wild type muscle microsomes was subjected to sucrose gradient fractionation and immunoblot analysis. In the wild type mice, the alpha 11.1, alpha 2delta , and a fraction of the gamma 1 subunits comigrate on the sucrose gradient (Fig. 6A), confirming that they are associated in a complex. Interestingly, a large fraction of the gamma 1 subunit does not appear to be in the complex and migrates in the lower fractions, and presumably this is developmentally regulated. In contrast, in the sucrose gradient fractions from the mdg muscle, the alpha 11.1 is not detected. However, the alpha 2delta and the gamma 1 subunits do not comigrate (Fig. 6A), suggesting that the presence of the alpha 11.1 subunit may be necessary for the gamma 1 subunit to associate with the calcium channel.


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Fig. 6.   gamma 1 interacts with the alpha 11.1. A, enriched skeletal muscle calcium channels from muscular dysgenesis (mdg) and 1-2-day-old wild type mice were subjected to sucrose gradient fractionation and immunoblot analysis. In the wild type mice, alpha 2 and gamma 1 comigrate, indicating the presence of an intact channel complex. In the mdg mice, gamma 1 and alpha 2 do not comigrate, indicating that alpha 11.1 may be required for gamma 1 to be associated with the channel. B, tsA201 cells were transiently transfected with the indicated constructs. Cell lysates were immunoprecipitated with an antibody to alpha 11.1. Aliquots of the lysate and the immunoprecipitated material were examined for the presence of the alpha 11.1 and the gamma 1EGFP. The alpha 11.1 immunoprecipitates with the gamma 1EGFP.

To examine if the gamma 1 subunit directly interacts with the alpha 11.1 subunit, subunit associations were examined in transiently transfected mammalian cells. TsA201 cells were transiently transfected with cDNAs encoding the alpha 11.1 subunit, the alpha 11.1 subunit, and the gamma 1EGFP or the gamma 1EGFP subunit alone. Cell lysates were subjected to immunoprecipitation using an antibody to the alpha 11.1 subunit. Aliquots of the lysate and the immunoprecipitated material were examined for the presence of the alpha 11.1 and gamma 1EGFP proteins (Fig. 6B). Our results show that in the cells transfected with the alpha 11.1 subunit, the alpha 11.1 subunit is immunoprecipitated, but the EGFP antibody does not detect any endogenous protein. Coexpression of alpha 11.1 and gamma 1EGFP allows coimmunoprecipitation, indicating complex formation. The gamma 1EGFP is not immunoprecipitated in the absence of the alpha 11.1 subunit, indicating specificity of interaction. These results confirm that the alpha 11.1 and the gamma 1 directly associate in the absence of the other subunits of the voltage-gated calcium channel.

Role of the First Extracellular Loop of gamma 1 in Subunit Interaction-- Our studies demonstrate that the first half of the gamma 1 subunit interacts with the calcium channel complex via the alpha 1 subunit. To examine the role of the first extracellular loop of the gamma 1 subunit in subunit interaction, a chimeric subunit was generated. This chimeric subunit (Fig. 7A) has the first extracellular loop of the gamma 1 subunit replaced by an extracellular loop of the unrelated four-transmembrane protein sarcospan (24) and EGFP at the C terminus. Transient transfection of tsA201 cells with the alpha 11.1 and gamma 1sspn and immunoprecipitation demonstrated that the two proteins associate (Fig. 7B). These results further support a role for the first two transmembrane domains of the gamma 1 subunit in subunit interaction.


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Fig. 7.   Role of the first extracellular loop in subunit interaction. A, schematic of the gamma 1sspnEGFP chimera. The first extracellular loop of gamma 1 is replaced by an extracellular loop of the unrelated protein sarcospan. B, tsA201 cells were transiently transfected with the indicated constructs. Cell lysates were immunoprecipitated with an antibody to alpha 11.1. Aliquots of lysate and immunoprecipitated material were examined for the presence of the alpha 11.1 and the gamma 1sspnEGFP. The alpha 11.1 immunoprecipitates with the gamma 1sspnEGFP. C, cell lysates from cells transfected with the gamma 1EGFP constructs and either treated with tunicamycin or untreated were immunoblotted with an antibody to GFP. Tunicamycin treatment inhibited N-linked glycosylation of gamma 1EGFP. D, tsA201 cells were transiently transfected with the indicated constructs and treated with tunicamycin. Cell lysates were immunoprecipitated with an antibody to alpha 11.1. Aliquots of the lysate and the immunoprecipitated material were examined for the presence of the alpha 11.1 and the gamma 1EGFP. The alpha 11.1 immunoprecipitates with the gamma 1EGFP in the absence of N-linked glycosylation.

The gamma 1 subunit has an N-linked glycosylation site in the first extracellular loop and is glycosylated in vivo. To examine the role of this glycosylation in the interaction of the gamma 1 subunit with the alpha 11.1 subunit, cells transiently transfected with the alpha 11.1 and gamma 1EGFP constructs, as described above, were treated with the aminoglycoside antibiotic tunicamycin, an inhibitor of N-linked glycosylation. Examination of cell lysates from treated and untreated cells indicated that the gamma 1EGFP existed in a smaller molecular weight form in tunicamycin-treated cells, indicating an absence of glycosylation (Fig. 7C). Furthermore, the gamma 1EGFP could be immunoprecipitated by an antibody to the alpha 11.1 protein, indicating that the lack of glycosylation does not inhibit the association of the alpha 11.1 subunit with the gamma 1 subunit (Fig. 7D).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The gamma  subunits of the voltage-gated calcium channels are the least characterized subunits of the calcium channels. In this study, we have dissected subunit interactions and domains mediating subunit interaction of the gamma 1 subunit with the other components of the skeletal L-type voltage-gated calcium channel. Moreover, we provide evidence for restricted subunit heterogeneity of the gamma  subunits. The voltage-gated calcium channels have a similar structure and subunit interactions, hence these studies have broad implications for the interactions of the gamma  subunits with the voltage-gated calcium channels (Fig. 8).


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Fig. 8.   Model of subunit interactions of the high voltage-gated calcium channels. The gamma  subunit is depicted as a four-transmembrane domain protein with predicted intracellular N and C termini. The first half of the gamma  subunit interacts with the alpha 1 subunit. The first extracellular loop contains charged residues and glycosylation sites. The interaction sites of the alpha 2delta (Gurnett et al. (9)) and the beta  subunit (Beta Interaction Domain-Pragnell et al. (19)) with the alpha 1 subunit have been defined previously.

The gamma  subunit was originally identified in skeletal muscle (12). In the recent past, a number of gamma  subunits that are expressed in a variety of tissues have been cloned (13-16), and their functional roles are only beginning to be revealed. In general, the gamma  subunits appear to be inhibitory (7, 22, 30, 34). The important physiological role for the gamma  subunit is emphasized by the epileptic phenotype in the stargazer mouse, a spontaneous mutant that lacks the gamma 2 subunit (13).

It is well known that the gamma 1 subunit modulates the inactivation kinetics and the current amplitude of the skeletal muscle voltage-gated calcium channels (21, 22). The gamma 1 subunit is not, however, required for maintaining the integrity of the channel complex or its targeting to the t-tubules. This is in sharp contrast to the distribution of the subunits in mdg mice and beta 1 null mice. alpha 2delta shows a punctate or diffused pattern in mdg mice (33), whereas alpha 11.1 is reduced or absent in beta 1 null mice (10). Recent studies have indicated that, unlike beta 1 (10), gamma 1 does not have a major role in membrane trafficking of the alpha 11.1 subunit as assessed by gating current measurements (21) or in EC coupling (35). Our results clearly demonstrate that the first half of the gamma 1 subunit that allows subunit interaction also allows the restoration of L-type currents in gamma 1 null muscle. Interestingly, despite the differences in L-type currents in the wild type and gamma 1 null mice, we have not observed any gross morphological changes in the muscle. Taken together, these data suggest that, at least in skeletal muscle, gamma 1 is predominantly involved in modulating the biophysical properties of the channel.

Subunit interactions are conserved across the subunits of the voltage-gated calcium channels as indicated by the presence of highly conserved interaction sites on the beta  subunit (19) and in vivo and in vitro subunit heterogeneity (17, 18). We have identified the interaction site of the gamma 1 with the calcium channel and predict that other gamma  subunits interact with alpha 1 subunits of the calcium channels similarly. Our studies indicate that the gamma  subunits may not be as capable of functional heterogeneity as beta  subunits, thereby restricting the number of possible subunit associations of the gamma  subunits with the voltage-gated calcium channels. However, the gamma  subunits have diverging homologies, with the gamma 6 subunit being the phylogenetically most closely related subunit to the gamma 1 subunit (15, 16). Hence, it is possible that the gamma  subunits might possess the ability to form heterogeneous complexes, albeit to a relatively limited extent as compared with the beta  subunits. This is also suggested by cell expression studies indicating the ability of the gamma 1 subunit to associate with the alpha 11.2 subunit (36), although it is associated with the alpha 11.1 in native tissue. The gamma 1 null mouse offers a good model system to test the ability of the other gamma  subunits to form heterogeneous complexes in an in vivo environment.

The t-tubule is a specialized organized structure continuous with the sarcolemma in the skeletal muscle. There is increasing evidence that the targeting of proteins to these structures involves determinants that are different from targeting to the sarcolemma (37). We have demonstrated the localization of the gamma 1 subunit in the t-tubules by microscopy. To our knowledge, this is the first report of localization of the gamma 1 subunit in the skeletal muscle. Interestingly, the gamma 1 subunit is localized to both the sarcolemma and the t-tubules, unlike the other subunits of the calcium channel, which are predominantly localized in the t-tubules (4). Whether this is an effect of overexpression of the protein is unclear. However, the localization of the protein at the plasma membrane in transfected mammalian cells suggests that the gamma 1 subunit contains the determinants for plasma membrane localization. In the future, it would be interesting to determine whether the ability of the gamma 1 subunit to localize to the t-tubules of the skeletal muscle, in addition to the plasma membrane/sarcolemma, is a result of its association with the subunits of the calcium channel or whether the protein has its own signals, like the alpha 11.1 subunit (38), to target it to the t-tubules, where it can then associate with the calcium channel complex.

We demonstrate that the first half of the gamma 1 mediates the interaction of the subunit with the rest of the calcium channel. This subunit interaction also allows the restoration of L-type calcium conductance in gamma 1 null myotubes. The absence of N-linked glycosylation of the gamma 1 subunit does not prevent the protein from associating with the alpha 11.1 subunit, suggesting that the N-linked glycosylation is not the predominant mediator of the interaction between the alpha 11.1 and gamma 1 subunits. Interestingly, the first extracellular loop of the gamma 1 subunit is relatively negatively charged (12) and has been suggested to be important in mediating the biophysical properties of the gamma 1 subunit (22). We demonstrate that the first extracellular loop of the gamma 1 subunit is not required for subunit interaction. Because the first half of the gamma 1 subunit interacts with the calcium channel, presumably via the transmembrane domains, the first extracellular loop would be predicted to be in close proximity to the alpha 11.1 subunit.

The subunit interactions of the gamma  subunits with the calcium channels have been contradictory, with suggestions of a requirement (7) or lack of requirement (36, 39) of the alpha 2delta subunit in the association of the gamma  subunit with the voltage-gated calcium channel. The studies on the mdg mice indicate that the alpha 2delta and gamma 1 subunits are not associated, thereby precluding a direct interaction between the two subunits. Interestingly, in the 1-2-day-old mice, there is a pool of gamma 1 subunit that is not associated with the calcium channel. The exact significance of this is unclear. It is possible that during development, there is an excess of the protein generated, and later gamma 1 that is not incorporated into the calcium channel is degraded. Alternatively, it is possible that during the early stages of muscle development, gamma 1 is associated with other proteins in addition to the calcium channel and involved in other unknown functions. Further studies are necessary to clarify the role of the gamma 1 subunits during early development. Our cell expression studies indicate that the gamma 1 subunit directly interacts with the alpha 1 subunit. This is consistent with studies that demonstrate a direct effect of the gamma 6 subunit on the alpha 13.1 protein in the absence of the other known calcium channel subunits (40). It is therefore likely that the requirement of the alpha 2delta subunit to mediate some of the electrophysiological properties contributed by the gamma  subunit reflect conformational changes rather than direct subunit interaction.

These studies provide valuable insights into the gamma  subunit interactions within the voltage-gated calcium channels. The past few years have seen the discovery of a number of different gamma  subunits and the confirmation of the four-subunit composition of the voltage-gated calcium channels. Understanding the subunit interactions of the gamma  subunits is an important step toward understanding the structural and functional subunit interactions within the voltage-gated calcium channels.

    ACKNOWLEDGEMENTS

We thank all the members of the Campbell lab for helpful discussions and critical reading of the manuscript and Melissa Hassebrock, Keith Garringer, Lindy McDonough, and Lindsay Williams for technical support. We thank the University of Iowa Diabetes and Endocrinology Research Center (supported by National Institutes of Health Grant DK25295) and the University of Iowa DNA Sequencing Core Facility. We also thank the University of Iowa Gene Transfer Vector Core, supported in part by the Carver Foundation and National Institutes of Health P30DK54759, for the generation of the adenoviruses.

    FOOTNOTES

* This work was supported by funding from the American Heart Association (to J. A. and C. A.), National Institutes of Health Grants RO1 AR 46448 (to R. C. and R. G.) and R01 HL 47053 (to R. C.), and the Muscular Dystrophy Association (to C. C. C. and V. A.).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.

|| Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 400 EMRB, Iowa City, IA 52242. Tel.: 319-335-7867; Fax: 319-335-6957; E-mail: kevin-campbell@uiowa.edu.

Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M208689200

    ABBREVIATIONS

The abbreviations used are: EC, excitation-contraction; WGA, wheat germ agglutinin; GFP, green fluorescent protein; EGFP, enhanced fluorescent green protein; aa, amino acid.

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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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