Complexes of the alpha 1C and beta  Subunits Generate the Necessary Signal for Membrane Targeting of Class C L-type Calcium Channels*

Tianyan Gao, Andy J. ChienDagger , and M. Marlene Hosey§

From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611

    ABSTRACT
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Abstract
Introduction
References

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha 1C subunit with different accessory beta  subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha 1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta  subunits also exhibited intracellular staining, co-expression of the alpha 1C subunit with either the wild-type beta 2a subunit, a palmitoylation-deficient beta 2a(C3S/C4S) mutant or three other nonpalmitoylated beta  isoforms (beta 1b, beta 3, and beta 4 subunits) resulted in the redistribution of both the alpha 1C and beta  subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha 1C was co-expressed with an N- and C-terminal truncated mutant beta 2a containing only the central conserved regions. However, when the alpha 1C subunit was co-expressed with an alpha 1beta interaction-deficient mutant, beta 2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta 2a that was unable to interact with the alpha 1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha 1C subunit with the largely peripheral accessory alpha 2delta subunit was ineffective in recruiting alpha 1C to the plasma membrane, while co-distribution of all three subunits was observed when beta 2a was co-expressed with the alpha 1C and alpha 2delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha 1 and beta  subunits.

    INTRODUCTION
Top
Abstract
Introduction
References

Voltage-dependent calcium channels are heteromultimeric complexes composed minimally of one of the various isoforms of the pore-forming alpha 1 subunit together with accessory beta  and alpha 2delta subunits (1, 2). The beta  subunits are very hydrophilic proteins and are localized toward the cytoplasmic face of the plasma membrane (1, 2). Four different beta  subunit genes have been cloned so far (3). All four different beta  subunits contain two conserved domains in the central region flanked by unique N and C termini (3). In contrast to the cytoplasmic orientation of the beta  subunit, the alpha 2delta subunit consists of the extracellular alpha 2 peptide disulfide-bonded to the membrane-spanning delta -peptide (1, 2). It has been shown that the accessory subunits play important roles in modulating calcium channel function (1). Co-expression of a beta  subunit with an alpha 1 subunit in heterologous expression systems results in an increase in the number of binding sites for drugs or toxins known to bind to the channels, an increase in peak current amplitude, and an increase in the number of channels at the cell surface (4-8). The increase in peak current amplitude observed upon beta  subunit co-expression is probably due to the increase in the number of plasma membrane-localized channels (5-7). The effects of the alpha 2delta subunit and the molecular events underlying alpha 2delta -mediated regulation of the channels are less well understood (1). Co-expression of the alpha 2delta subunit with the cardiac alpha 1C subunit resulted in increased peak current amplitude and altered channel kinetics in Xenopus oocytes (9, 10). However, kinetic changes were not observed in other studies (11, 12).

Recently, we and others reported that native L-type calcium channels in rabbit cardiac myocytes (class C L-type channels) are minimally composed of an alpha 1C subunit, a beta 2 subunit, and an alpha 2delta subunit (13, 14). Co-localization of the calcium channel beta  and alpha 2delta subunits with the alpha 1 subunits in the T-tubule membranes of heart cells was observed (13). Direct interactions between alpha 1 and beta  subunits (15, 16) or between alpha 1 and alpha 2delta subunits (17) have been identified, and the regions responsible for subunit interaction have been partially mapped out in each subunit (15-18). However, more biochemical studies are required to further understand the modulatory effects of the alpha 2delta and beta  subunits.

Little is known about the targeting signals that direct calcium channels to the plasma membrane. We have recently demonstrated that the rat beta 2a isoform is palmitoylated, while other beta  isoforms, including the beta 1b, beta 3, and beta 4 subunits, are not (5). The palmitoylation sites have been mapped to Cys3 and Cys4 in the N terminus of the rat beta 2a subunit (5). Palmitoylation appears to play a role in the targeting of the beta 2 subunit to the plasma membrane when it is expressed in the absence of an alpha 1 subunit (19). Palmitoylation-deficient beta 2a mutants and other nonpalmitoylated beta  isoforms were localized intracellularly when expressed in the absence of other channel subunits (19). In addition, palmitoylation appears to confer unique regulatory properties to the rat beta 2a subunit (20). However, it remains unclear whether palmitoylation is an important signal for targeting of channel complexes. Here, we further investigated the role of beta  subunits in plasma membrane targeting of calcium channel complexes. To identify the region in beta  subunits required for targeting, we have expressed the alpha 1C subunit with different wild-type and mutant beta  subunits in HEK-tsA201 cells and assessed the localization of channel subunits using confocal microscopy and whole-cell radioligand binding studies. Since the membrane and extracellular orientation of the alpha 2delta subunit might suggest a role in targeting other channel subunits to the membrane, the effect of the alpha 2delta subunit in membrane targeting of the channel complexes was also studied.

    EXPERIMENTAL PROCEDURES

Materials-- All reagents were obtained from general sources unless otherwise noted. The calcium channel subunit-specific antibodies used in this study including Card C, beta GEN, and anti-beta 2 antibodies were described previously (4, 5). The expression vector used with the alpha 2delta subunit, pMT21alpha 2delta , was as described (13). Generation and expression of several mutant beta  subunits including beta 2aCys-, beta 2aIFP, and beta 2aBID- were described previously (19). The monoclonal anti-alpha 2 antibody was a generous gift from Drs. Sylvie Vandaele and Michel Lazdunski (21).

Generation of a Mutant beta  Subunit Expression Construct-- To construct a truncation mutant of the beta 2a subunit, beta 2a-(17-411) (beta 2aDelta NC), the 5'-untranslated region and initiator ATG were amplified by the polymerase chain reaction using the following oligonucleotide primers: 5'-AAGCTTAGCAACAGCTCGGTCAGG-3' (plus strand) and 5'-GGATCCCATGAAGAGGTGGCAGGACG-3' (minus strand). This initial fragment was cloned as a HindIII-BamHI fragment into a modified version of pCR3 (Invitrogen) containing a C-terminal KT3 epitope tag (4) in frame with a BamHI site. Subsequently, a fragment encoding amino acids 17-411 of beta 2a was amplified by polymerase chain reaction using the following primers: 5'-GGATCCGCAGACTCCTACACCAGC-3' (plus strand) and 5'-AGATCTGTGGGTGGCCTTCCAGTACGC-3' (minus strand). The beta 2a-(17-411) fragment was cloned into the BamHI site of the previously described vector containing the beta 2a initiator ATG and KT3 epitope tag as a BamHI-BglII fragment; orientation was confirmed by restriction endonuclease analysis.

Cell Culture and Transfection-- HEK-tsA201 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin at 37 °C in 5% CO2. Transient expression of different calcium channel subunits in HEK-tsA201 cells was carried out using the calcium phosphate precipitation method (4).

Immunofluorescence Staining and Confocal Microscopy-- Transiently transfected HEK-tsA201 cells were stained with calcium channel subunit-specific antibodies following the procedures described previously (4, 19). Briefly, the transfected cells were fixed in precooled (-20 °C) methanol/acetone (1:1) for 5-10 min at 4 °C and followed by incubating with labeling buffer (1% bovine serum albumin in phosphate-buffered saline, pH 7.4) to block nonspecific binding. Different primary antibodies were diluted in labeling buffer and incubated with cells for 1-2 h at room temperature. The secondary antibodies including fluorescein isothiocyanate-conjugated goat anti-rabbit IgG, Alexa 488-conjugated goat anti-rabbit IgG, and tetramethylrhodamine isothiocyanate-conjugated rabbit anti-goat IgG (Molecular Probes, Inc., Eugene, OR) were used subsequently. The cellular distributions of the expressed channel proteins were visualized using a Zeiss LSM-10 laser scanning microscope.

Immunoprecipitation and Immunoblotting-- Transfected tsA201 cells were homogenized, and crude membrane fractions were prepared as described previously (4). For co-immunoprecipitation of alpha 1 and beta  subunits, membrane particulate fractions were solubilized in solubilization buffer (50 mM Tris, pH 7.4, 5 mM EDTA, 5 mM EGTA, 0.4 M NaCl, 1% Triton X-100, and 0.1% SDS containing protease inhibitors (4)). Solubilized proteins were immunoprecipitated overnight with agitation at 4 °C using beta GEN antibodies coupled to protein G-Ultralink resin (Pierce). Immunoprecipitates were washed with solubilization buffer 3-5 times and eluted with SDS sample buffer. Immunoblotting procedures were performed as described previously (4). Detection of immunoreactive bands was performed using either enhanced chemiluminescence (Pierce) or colorimetric enhanced diaminobenzidine substrate reaction (Pierce).

Intact Cell Radioligand Binding-- To detect membrane localization of L-type calcium channels, ligand binding experiments were performed using the dihydropyridine (DHP)1 radioligand (+)-[3H]PN200-110 with intact cells transfected with different combinations of the channel subunits. The experimental procedures were as described previously (4). Scatchard analyses were performed to assess Bmax and Kd values.

    RESULTS

Palmitoylation of the beta 2a Subunit Is Not Necessary for Functional Channel Formation-- Previously, we had demonstrated that co-expression of the rat beta 2a subunit with the alpha 1C subunit resulted in an increase in the number of functional channels at the cell surface as assessed by immunohistochemical and electrophysiological analyses (4). We and others subsequently confirmed this finding by electrophysiological measurements of whole-cell charge movement (5-7). Since the beta 2a subunit is a palmitoylated protein (5), it was possible that palmitoylation was playing a role in targeting channels to the plasma membrane. However, co-expression of either the wild-type beta 2a subunit or the palmitoylation-deficient beta 2a(C3S/C4S) subunit with the alpha 1C subunit resulted in an increase in the amount of measured whole-cell charge-movement, suggesting that palmitoylation was not required for the formation of channel complexes (5). In order to obtain further insight into the role of palmitoylation of the beta 2 subunit in the formation and membrane targeting of alpha 1C complexes, transiently transfected cells expressing the alpha 1C subunit alone or the alpha 1C subunit in combination with either the wild-type beta 2a or the mutant beta 2a(C3S/C4S) subunits were analyzed biochemically as well as by confocal immunofluorescent microscopy.

Membrane particulate fractions from transfected cells expressing the alpha 1C subunit in combination with either the beta 2a or the beta 2a(C3S/C5S) subunit were solubilized as described under "Experimental Procedures." Following immunoprecipitation of solubilized proteins with the beta GEN antiserum, immunoblotting was used to detect the presence of the alpha 1C and beta 2a proteins in the immunoprecipitated pellets. Immunostaining of the immunoprecipitates with the Card C antiserum revealed that the alpha 1C subunit co-purified with both the beta 2a and beta 2a(C3S/C4S) subunits (Fig. 1A, top), suggesting that the loss of palmitoylation in the beta 2a(C3S/C4S) mutant did not noticeably disrupt interactions between the alpha 1C and beta 2a subunits. The presence of the beta 2a and beta 2a(C3S/C4S) subunits in the immunoprecipitated pellets was confirmed by immunoblotting with the beta 2a antiserum (Fig. 1A, bottom).


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Fig. 1.   Palmitoylation of the beta  subunit was not necessary for the targeting of functional channels to the cell surface upon beta  subunit co-expression. Transiently transfected cells expressing the alpha 1C subunit alone or in combination with either the wild-type beta 2a subunit or the palmitoylation-deficient beta 2a(C3S/C4S) mutant were analyzed by both co-immunoprecipitation and immunohistochemical studies. A, solubilized membrane fractions from transfected cells were immunoprecipitated with the beta GEN antiserum. Immunoblotting with the beta 2a antiserum (bottom) was used to confirm the immunoprecipitation of beta 2a protein. Immunoblotting with the Card C antiserum (top) was used to detect the amount of alpha 1C protein, which was co-immunoprecipitated, presumably through interaction with the beta 2a subunit. B represents the typical Card C staining pattern observed in transiently transfected cells expressing the alpha 1C alone. No discernible immunostaining is seen at the cell surface. C, staining of alpha 1Cbeta 2a cells with the Card C antibody (left) revealed punctate alpha 1C distribution at and/or near the plasma membrane, similar to previously published results (4). Similarly, the staining pattern of the beta 2a antibody revealed a punctate distribution of the beta 2a subunit in these co-transfected cells. This confocal image section was taken from near the top surface of the cell. D, staining of alpha 1Cbeta 2a(C3S/C4S) cells with the Card C antibody (left) also revealed punctate alpha 1C distribution along the cell surface (top view). Additionally, immunostaining with the beta 2a antibody revealed a punctate localization of the beta 2a(C3S/C4S) protein at the plasma membrane as well.

In confocal microscopy studies, cells expressing the alpha 1C subunit alone exhibited a perinuclear staining pattern (Fig. 1B, left; the phase image is shown on the right), while co-expression of the alpha 1C subunit with the beta 2a subunit resulted in a striking change of the alpha 1C subunit staining to a punctate pattern at the plasma membrane (Fig. 1C, left). Both of these findings are consistent with previous results (4). Interestingly, when co-expressed with the alpha 1C subunit, the staining pattern of the beta 2a subunit also changed to a more punctate and less continuous pattern (Fig. 1C, right) along the cell surface compared with the continuous membrane staining of cells expressing the beta 2a subunit alone that we observed previously (19). It was of interest to determine if a similar or different pattern of staining would be observed when the alpha 1C subunit was co-expressed with the palmitoylation-deficient beta 2a(C3S/C4S) subunit. Co-expression of the alpha 1C subunit with the beta 2a(C3S/C4S) mutant resulted in punctate staining of the alpha 1C subunit at the cell surface (Fig. 1D, left panel). Notably, co-expression with the alpha 1C subunit also led to redistribution of the beta 2a(C3S/C4S) protein to the plasma membrane (Fig. 2D, right), despite the fact that this mutant protein, when expressed in the absence of the alpha 1C subunit, was localized intracellularly (19). These results support the hypothesis that the multimerization of channel subunits, which likely occurs during or shortly after synthesis of the individual peptides, allows the proper folding and assembly of channel complexes and facilitates their transport to the cell surface. From these results, as well as previously reported electrophysiological results (5), the lack of palmitoylation of the beta 2a subunit did not appear to affect subunit interactions that are critical for channel formation and targeting.


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Fig. 2.   Redistribution of calcium channel alpha 1C subunits by co-expression with different beta  subunits. The alpha 1C and different beta  subunits were co-expressed in tsA cells. The cells transiently transfected with alpha 1Cbeta 1b (A), alpha 1Cbeta 3 (B), or alpha 1Cbeta 4 (C) were immunofluorescently stained with the Card C antibody to reveal the expression patterns of the alpha 1C subunits, and visualization was with confocal microscopy. A phase-contrast image of alpha 1Cbeta 4-transfected cells was included as an example to show the plasma membrane outlines of the cells (C). Punctate channel clusters were observed along cell membranes in all three types of transfected cells, and the distribution patterns were similar to those seen in Fig. 1, C and D.

The alpha 1C Subunits Were Redistributed to the Plasma Membranes by Co-expression with Different beta  Isoforms-- To test whether the three other beta  isoforms can form complexes with and cause membrane targeting of the alpha 1C subunit, we co-expressed the alpha 1C subunit with the beta 1b, beta 3, or beta 4 subunits in tsA201 cells. The subcellular localization pattern of the alpha 1C subunit was revealed by immunostaining with the Card C antibody. Punctate staining of alpha 1C subunit clusters was observed along plasma membranes in alpha 1Cbeta 1b-, alpha 1Cbeta 3-, and alpha 1Cbeta 4-transfected cells (Fig. 2). A phase-contrast image of alpha 1Cbeta 4-transfected cells was used as an example to confirm the plasma membrane outlines of the cells (Fig. 2C, right). Although the beta 1b, beta 3, and beta 4 subunits were nonpalmitoylated and were intracellularly localized when expressed alone (5, 19), all three beta  subunits were able to cause redistribution of the alpha 1C subunit from a perinuclear location to the plasma membrane. These results suggested that each beta  subunit was able to form complexes with the alpha 1C subunit in a manner similar to that observed with the beta 2a palmitoylation-deficient mutant, confirming that the palmitoylation of the beta  subunits was not required for membrane targeting of calcium channel complexes when co-expressed with the alpha 1C subunit.

The Conserved Core Region of beta  Subunits Is Sufficient for Membrane Targeting of Channel Complexes-- The finding that each beta  subunit caused a similar redistribution of the alpha 1C subunit to the plasma membrane suggested that a structural domain common to each beta  subunit might be important. All four different beta  subunits discovered to date (including numerous splicing variants) contain a conserved central region with two highly homologous domains (3). Within this region, a site that mediates interaction with various alpha 1 subunits has been identified and termed the beta -interaction domain (BID) (16, 22). However, it has not been demonstrated whether the conserved core region of the beta 2 subunit is sufficient for membrane targeting of the alpha 1Cbeta 2 complex. To test this possibility, an N- and C-terminal truncation mutant of the rat beta 2a subunit, beta 2aDelta NC, containing only the conserved core region, was created and transfected into HEK-tsA cells. An intracellular staining pattern of this minimal beta  subunit was observed when this truncation mutant was expressed alone (data not shown). This finding was consistent with the fact that the N-terminal palmitoylation sites (membrane-anchoring sites) were deleted from this truncation mutant (19). To study the membrane targeting function of beta 2aDelta NC, the alpha 1C and beta 2aDelta NC subunits were co-expressed in tsA cells, and the expression pattern of the alpha 1C subunit was revealed using the Card C antibody. Clusters of alpha 1C subunits were observed as bright punctate spots on the cell surface, as shown in the confocal image generated from a top section of the cell (Fig. 3, top panel). A confocal image taken from the middle section of the cell showed punctate formation of channel complexes along the cell plasma membranes (Fig. 3, top panel). These staining patterns were similar to those seen in Figs. 1 and 2. The ability of the beta 2aDelta NC subunit to allow targeting of the alpha 1Cbeta 2 complex to the cell plasma membranes indicated that the conserved core region of beta  subunits was sufficient for the targeting of the alpha 1C·beta complexes. Taken together, these results suggested that the membrane targeting observed in the presence of all beta  subunits can be achieved with only the conserved region. However, the unique electrophysiological properties of calcium channels that have been observed upon co-expression of different beta  isoforms were probably determined by distinctive N- and C-terminal regions (1, 20).


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Fig. 3.   The conserved core region of the beta  subunits is required and sufficient for membrane targeting of channel complexes. The beta 2aDelta NC subunit, a truncation mutant of beta 2a, was co-expressed with the alpha 1C subunit in tsA cells. The expression pattern of the alpha 1C subunits was revealed by the Card C antibody using confocal microscopy. The confocal image shown in the top panel was obtained from the top surface of the cell, and the channel clusters were observed as bright round spots on the cell surface. The image shown in the lower panel was obtained from a middle section across the cell, and the staining pattern was punctate along the cell plasma membrane. The staining patterns of the alpha 1C subunit shown here are similar to those observed in Figs. 1 and 2.

A Mutation of the BID Disrupted Channel Targeting and Subcellular Localization of the beta 2a Subunit-- The results presented so far suggested that the signal for membrane targeting of the alpha 1C and beta  subunits generated upon formation of alpha 1C·beta complexes, since membrane targeting was achieved in most instances with subunits that exhibited an intracellular distribution when expressed alone. If this is true, then mutations that disrupt subunit interaction should disrupt membrane targeting. Electrophysiological studies in Xenopus oocytes, as well as studies with fusion proteins, have suggested that the BID in the conserved domains of beta  subunits is critical for alpha beta subunit interaction (16). Mutation of Pro237 to Arg in the BID of the beta 1b subunit appeared to completely eliminate alpha 1beta interaction (16). However, studies with full-length beta  and full-length alpha 1C subunits have not been performed to determine if this BID region was also critical for channel targeting to the plasma membrane. Therefore, site-directed mutagenesis was used to create a mutation at Pro234 (analogous to Pro237 of the beta 1b subunit) in the beta 2a subunit. The wild-type beta 2a subunit or the beta 2a(P234R) mutant was transiently expressed in tsA201 cells in combination with the alpha 1C subunit and assessed for subunit interactions. Membrane particulate fractions from these cells were solubilized as described under "Experimental Procedures," and the soluble fractions were subsequently immunoprecipitated with the beta GEN antibody. Immunoblotting with Card C and beta 2a antisera was used to detect the presence of the alpha 1C and beta 2a subunits, respectively, in the immunoprecipitated pellets. Immunoprecipitation of the wild-type beta 2a subunit from alpha 1C cells resulted in co-purification of the alpha 1C subunit in the immunoprecipitated pellet (Fig. 4A, top left). By contrast, immunoprecipitation of the beta 2a(P234R) protein did not result in co-purification of the alpha 1C subunit. The alpha 1C subunit was seen in both the input and the flow-through lanes from alpha 1Cbeta 2a(P234R) cells (Fig. 4A, right), indicating that the absence of co-immunoprecipitation was not due to lack of alpha 1C protein but rather to a lack of interaction between the alpha 1 and beta  proteins. Transiently transfected alpha 1Cbeta 2a(P234R) cells were fixed and immunohistochemically stained with the Card C and beta 2a antibodies (Fig. 4B). The alpha 1C protein was localized to perinuclear regions in these cells (Fig. 4B, left), similar to the distribution observed in cells expressing the alpha 1C subunit alone (see Fig. 1B). Similarly, the beta 2a(P234R) subunit exhibited a diffuse intracellular staining pattern (Fig. 4B, right) indistinguishable from that of beta 2a(P234R) expressed alone (19). Thus, disruption of the interaction between the alpha 1 and beta  subunits caused by the mutation in BID also disrupted membrane targeting of channels in these cells. Alternatively, mutation of Pro234 may have affected subcellular localization and channel targeting through global disruption of the beta 2a protein structure.


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Fig. 4.   Mutation of the BID prevented channel targeting and altered the subcellular localization of the beta 2a protein. A, transiently transfected cells expressing the alpha 1C subunit in combination with either the wild-type beta 2a subunit or the beta 2a(P234R) mutant were used to assess the effect of this mutation on the ability of the alpha 1C and beta 2a subunits to co-purify as a complex. Solubilized fractions from transfected cells were immunoprecipitated with the beta GEN antiserum. Subunits were detected by immunoblotting with the Card C (upper left) and beta 2a (lower left) antibodies. The input and flow-through fractions (6% of each) from alpha 1Cbeta 2a(P234R) cells were immunoblotted with the Card C antiserum (right), indicating that the lack of alpha 1C protein in the beta GEN immunoprecipitate was not due to the absence of alpha 1C protein. B, transiently transfected alpha 1Cbeta 2a(P234R) cells were immunohistochemically stained with the Card C and beta 2a antibodies and analyzed by confocal immunofluorescence microscopy. Card C staining in these cells was perinuclear (left), similar to the distribution pattern observed in cells expressing the alpha 1C subunit alone (Fig. 1B). Interestingly, the staining by the beta 2a antibody revealed a diffuse intracellular distribution of the beta 2a(P234R) protein (right).

An SH3 Motif Mutation of beta 2a Disrupted Channel Targeting and Interaction with the alpha 1C Subunit-- An SH3-like motif was found in the conserved region of all four beta  subunits (19). We have reported that a triple point mutation in this SH3 motif (beta 2a(I115A/F117A/P119L), or beta 2aIFP) of the beta 2a subunit resulted in a marked reduction of palmitoylation and an intracellular rather than membrane localization of the protein (19). The mutated amino acids correspond to residues previously identified to play key roles in the SH3 domain of Src (23, 24). Here, we investigated the effect of the SH3 domain on membrane targeting of the channel complexes. The SH3 motif mutant, beta 2aIFP, and the alpha 1C subunits were co-expressed in tsA cells, and expression patterns of both the alpha 1C and mutant beta 2aIFP subunits were visualized using confocal microscopy. Interestingly, co-expression of beta 2aIFP with alpha 1C did not result in redistribution of the channels. As shown in Fig. 5A, both the alpha 1C (left) and beta 2aIFP (right) subunits remained in cytoplasmic regions. The staining patterns of both subunits were indistinguishable from those observed with either subunit expressed alone in cells (19).


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Fig. 5.   An SH3-motif mutant disrupted the interaction between the alpha 1C and beta 2a subunits and failed to target the channels to the plasma membrane. A, the alpha 1C and an SH3 mutant of beta 2a, beta 2aIFP, were co-transfected into tsA cells. The expression patterns of both the alpha 1C and mutant beta 2a subunits were revealed by immunofluorescent staining with Card C (left) or the beta 2 antibody (right), respectively. The confocal images showed intracellular staining patterns for both subunits. B, the crude membrane fractions were prepared from alpha 1Cbeta 2a- or alpha 1Cbeta 2aIFP-co-transfected cells, and solubilized proteins were immunoprecipitated with the beta GEN antibody. The immunoprecipitates were analyzed with SDS-polyacrylamide gel electrophoresis and immunoblotting. The wild-type beta 2a and beta 2aIFP mutant subunits immunoprecipitated by beta GEN were detected with the beta 2 antibody on the immunoblot (bottom). However, the alpha 1C subunit was only co-immunoprecipitated with the wild-type beta 2a subunits (top, lane 1, detected with the Card C antibody), but not with the beta 2aIFP mutant subunits (top, lane 2). Lane 3 shows the presence of the alpha 1C subunits in the flow-through fraction from immunoprecipitation of the alpha 1Cbeta 2aIFP cells.

To test whether lack of a membrane targeting function of beta 2aIFP is due to disruption of alpha 1beta interaction, association between alpha 1C and beta 2aIFP was analyzed using immunoprecipitation. Membrane particulate fractions were prepared from alpha 1Cbeta 2aIFP-co-transfected cells, and solubilized proteins were immunoprecipitated with the beta GEN antibody. As a positive control, co-immunoprecipitation of the alpha 1C and wild-type beta 2a subunits was easily detected (Fig. 5B, lane 1). However, the alpha 1C subunit did not co-immunoprecipitate with the beta 2aIFP (Fig. 5B, lane 2) subunits, suggesting that the interaction between these two subunits was largely impaired. The alpha 1C subunit in alpha 1Cbeta 2aIFP-co-transfected cells was detected in the supernatant fraction of the beta GEN immunoprecipitation (Fig. 5B, lane 3), indicating that lack of alpha 1Cbeta 2aIFP co-precipitation was not due to lack of alpha 1C expression. Taken together, these results suggested that the SH3 domain of the beta  subunits may play a role in alpha 1beta interaction. Alternatively, the mutations may have disrupted post-translational modifications or proper folding of the beta  subunit and caused a loss in normal function, although the mutant beta  subunit was able to be recognized by the beta 2 and beta GEN antibodies, suggesting a lack of global disruption of protein structure.

DHP Binding and Plasma Membrane Formation of L-type Calcium Channels-- To confirm that the immunofluorescence staining of the channel subunits observed along cell surfaces represented functional calcium channels at the plasma membrane, whole-cell DHP binding experiments were performed using the radiolabeled antagonist (+)-[3H]PN200-110. Since (+)-[3H]PN 200-110 is a hydrophobic ligand, the specific binding obtained with intact cells should reflect binding to calcium channels located on the plasma membrane. Different combinations of the calcium channel subunits, including wild-type alpha 1Cbeta 2a, alpha 1Cbeta 2a(C3S/C4S), alpha 1Cbeta 2aDelta NC, alpha 1Cbeta 2aIFP, and alpha 1Cbeta 2aBID-, were transfected into tsA cells. Whole-cell binding experiments were performed, and results were subjected to Scatchard analysis.

For cells transfected with wild-type alpha 1Cbeta 2a, alpha 1Cbeta 2a(C3S/C4S), or alpha 1Cbeta 2aDelta NC, saturable DHP binding was observed in all three cases. The Bmax and Kd values were between 0.5-1.2 pmol/mg protein and 0.2-0.6 nM, respectively, from four independent experiments, and no appreciable differences among these subunit combinations were observed (Table I). In contrast, no saturable binding was obtained from cells transfected with either alpha 1Cbeta 2aIFP or alpha 1Cbeta 2aBID-. These DHP binding results were in agreement with the expression patterns observed in the immunofluorescence staining studies, suggesting that the functional L-type calcium channels were targeted to the cell plasma membrane in alpha 1Cbeta 2a-, alpha 1Cbeta 2a(C3S/C4S)-, and alpha 1Cbeta 2aDelta NC-transfected cells. However, when plasma membrane expression of channel complexes was not observed in the immunostaining studies, as for the cases of alpha 1Cbeta 2aIFP and alpha 1Cbeta 2aBID-, no specific DHP binding was obtained as well.

                              
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Table I
Whole-cell DHP binding in tsA cells transfected with calcium channel subunits
Different combinations of calcium channel subunits were co-transfected into tsA cells, and whole-cell binding experiments were performed using the radioligand (+)-[3H]PN200-110. Results shown were from four independent experiments. The Bmax and Kd values were determined using Scatchard analysis. The differences in Bmax values probably resulted from different transfection efficiencies. Since no saturable binding was obtained in cells transfected with alpha 1Cbeta 2aBID- and alpha 1Cbeta 2aIFP subunits, Kd values were not determined (ND). No significant differences were observed in the binding parameters between the cells transfected with different channel subunits as indicated by statistical analyses. The values are expressed as mean ± S.E.

A Different Distribution Pattern of the alpha 1C Subunit Was Obtained upon Co-expression with the alpha 2delta Subunit-- Recently, it has been demonstrated that alpha 1 subunits can associate with alpha 2delta subunits through direct interactions between the transmembrane domains of alpha 1 and the delta  peptide and the extracellular regions of alpha 1 and the alpha 2 peptide (17, 18). To address the possibility that the alpha 2delta subunit also can play a role in the membrane targeting of calcium channel complexes, we transiently expressed the alpha 1C and alpha 2delta subunits in tsA cells. Card C and a monoclonal anti-alpha 2 antibody were used to reveal the expression patterns of the alpha 1C and alpha 2delta subunits, respectively. When the alpha 2delta subunit was expressed alone in the cells, the majority of the protein localized to the plasma membrane and exhibited smooth rather than punctate staining (Fig. 6A). We also observed some faint intracellular staining of alpha 2delta , which may reflect newly synthesized or partially processed proteins. This finding is different from that previously obtained by Brice et al. (25), who reported an intracellular staining pattern of alpha 2delta subunits when expressed in COS-7 cells. This inconsistency may be explained by different glycosylation and processing machinery in specific cells, since the highly glycosylated alpha 2delta subunit may require proper processing and glycosylation to be inserted into the plasma membrane (26). Co-expression of the alpha 1C and alpha 2delta subunits in tsA cells did not allow significant targeting of the alpha 1C subunit to the plasma membrane (Fig. 6B). Only a very small portion of the alpha 1C subunits localized to the cell membrane (Fig. 6B, indicated by arrows), while the majority of the alpha 1C subunits remained intracellular (Fig. 6B). This result suggested that the alpha 2delta subunit was unable to mimic the targeting function observed with the beta  subunits. In addition, since the alpha 2delta subunit itself did localize to the plasma membrane, the results suggest that complex formation between alpha 1C and alpha 2delta is less efficient than between alpha 1C and beta  subunits. This suggestion is consistent with the fact that the alpha 2delta subunit-mediated regulation of channel function is less dramatic than that observed with the beta  subunits (6, 9, 10).


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Fig. 6.   Co-expression of alpha 2delta with alpha 1C subunits did not redistribute the alpha 1C subunits into clusters along the cell surface. A and B are confocal images showing expression patterns of the alpha 2delta and alpha 1C subunits. A, the cells transfected with the alpha 2delta subunits alone were stained with a monoclonal anti-alpha 2delta antibody. A plasma membrane expression pattern was observed. B, the alpha 1C and alpha 2delta subunits were co-transfected into tsA cells, and the cells were stained with Card C to visualize the expression pattern of the alpha 1C subunits. A faint plasma membrane staining was obtained (indicated by a right arrow); however, the majority of the alpha 1C subunits remained intracellular. C, the cells co-transfected with alpha 1Cbeta alpha 2delta subunits together, and stained with Card C, beta 2, and alpha 2 antibodies to reveal the expression pattern of each subunit. Similar punctate plasma membrane staining patterns were observed for all three subunits.

To test targeting of the channel subunits when all three subunits were expressed, we co-expressed alpha 1C, beta 2a, and alpha 2delta subunits together into tsA cells. All three subunits localized to the plasma membrane as shown in Fig. 6C, and similar punctate staining patterns along the plasma membrane were observed for all three subunits. Interestingly, the punctate staining pattern for the alpha 2delta subunit in alpha 1beta alpha 2delta -co-transfected cells was different from the smooth staining seen in the cells transfected with the alpha 2delta subunit alone (Fig. 6, compare C (right) with A). The punctate pattern of the alpha 2delta subunit when co-expressed with other subunits is consistent with channel complex formation. In addition, saturable DHP binding was obtained in alpha 1Cbeta alpha 2delta -co-transfected cells, confirming that functional channels were targeted to the plasma membrane (data not shown). Moreover, when the alpha 1Cbeta alpha 2delta -co-transfected cells were co-stained with antibodies for the alpha 1C and alpha 2delta subunits or the beta 2a and alpha 2delta subunits, overlays of confocal images indicated that all three subunits were co-localized along cell plasma membranes (data not shown). These results further demonstrated the importance of the beta  subunits in membrane targeting, since co-expression of the beta  subunit was necessary for the proper formation of channel complexes composed of all three channel subunits (compare Fig. 1C with Fig. 6C).

    DISCUSSION

We previously demonstrated that co-expression of calcium channel beta  subunits with alpha 1 subunits resulted in formation of clusters of channels along the cell plasma membranes (4). In the present study, we further investigated the structural determinants of the beta  subunits for membrane targeting. Although palmitoylation was required for membrane localization of the rat beta 2a subunit when this subunit was expressed in the absence of other subunits in tsA cells (19), the results presented here clearly demonstrated that the palmitoylation state of the beta  subunits was not a major determining factor for plasma membrane targeting of channel complexes when beta  subunits were co-expressed with the alpha 1C subunit. We examined the expression patterns of channel complexes in alpha 1Cbeta 2a-, alpha 1Cbeta 2aCys--, alpha 1Cbeta 1b-, alpha 1Cbeta 3-, and alpha 1Cbeta 4-co-transfected cells, and punctate clusters of channels were observed at the plasma membrane in all cases despite the fact that only the wild-type beta 2a was palmitoylated. In addition, we demonstrated that the central conserved core region of the beta  subunits was necessary and sufficient for membrane targeting of alpha 1C·beta complexes. Thus, the N-terminal variable regions containing the palmitoylation sites in the rat beta 2a subunit were not required for the targeting and punctate distribution of channel complexes. However, several mutations in the conserved region of the beta 2a subunit that disrupted alpha 1beta interactions resulted in a failure to target the alpha 1C and beta  subunits to the plasma membrane. Taken together, our results suggested that a functional interaction between the alpha 1C and the conserved domains of the beta  subunits is required for membrane targeting of the class C L-type channels. Interestingly, our results demonstrated that the targeting we observed also was responsible for movement of the beta  subunits to the plasma membrane. For example, although the beta 2a(C3S/C4S) and the beta 2aDelta NC mutants were localized intracellularly when expressed alone (19), upon complex formation with the alpha 1C subunit, both of these mutant subunits redistributed to the plasma membrane, and exhibited a punctate staining pattern similar to the alpha 1C subunit. Since neither alpha 1C nor these mutant beta  subunits, nor the wild-type beta 1b, beta 3, and beta 4 subunits, are able to target to the plasma membrane when expressed alone, these results strongly suggest that the signals for membrane targeting are contained in the complex of alpha 1C·beta subunits rather than in either subunit alone. Thus, it is not surprising that mutations that disrupt complex formation, such as the beta 2aBID- and beta 2aIFP mutants, disrupt membrane targeting. Conceivably, a membrane targeting "element" is formed upon the association of the alpha 1C and beta  subunits. This membrane targeting element may involve a region in either one of the subunits that is exposed due to subunit association or may involve an intersubunit domain.

An interesting finding from our present study is that co-expressed alpha 1Cbeta complexes formed punctate clusters along cell plasma membranes. Channel clustering has been identified for several channel types. For example, shaker K channels and NMDA receptors are found to form clusters along cell membranes through association with PDZ-domain containing proteins (27, 28). Our results suggested that either the alpha 1C or the beta  subunits, or alternately, complexes of alpha 1C and beta  subunits, may form secondary interactions with other cellular proteins such as PDZ-domain-containing proteins, and this secondary protein-protein interaction may result in channel clustering at specific plasma membrane locations (hot spots). However, the mechanisms underlying calcium channel clustering are not clear at this point. Of interest, the association between the alpha 1C and alpha 2delta subunits did not appear to be sufficient or strong enough to target the alpha 1C subunit to the plasma membrane, although alpha 1alpha 2delta interactions have been described (17, 18). Alternatively, the association of alpha 1 and alpha 2delta may require the presence of the beta  subunit, since the conformation assumed by the alpha 1·beta complex may allow better recruitment of alpha 2delta to the complex. When alpha 1beta alpha 2delta subunits were co-expressed in cells, channel clusters containing all three subunits were observed along plasma membranes. Our present findings may also explain previous electrophysiological results in mammalian cells that demonstrated that the effect of the alpha 2delta subunit in augmenting calcium channel currents was much less significant compared with that of the beta  subunits, while the effects of beta  and alpha 2delta were synergistic (12).

We have previously identified an SH3-like motif that is present in all four beta  subunits known to date (19). SH3 domains have been shown to mediate protein-protein interaction in many important signaling processes (29). The SH3 motif of the beta  subunits is in the conserved core region and about 100 amino acids upstream of the BID domain (19). There are two possibilities that may account for our finding that mutations in the SH3 motif of beta  subunits disrupted interaction with the alpha 1C subunit. First, since the SH3 motif is adjacent to the BID region in the beta  subunits, mutations in the SH3 domain may interfere with direct binding between alpha 1 and the BID domain of beta  subunits. Alternatively, the SH3 motif may be a structurally important determinant of the beta  subunits, and mutations in this region may disrupt the important protein-protein interactions or important structural domains in the beta  subunits. Our previous findings that the palmitoylation level of beta 2aIFP was significantly decreased also suggested the structural importance of the SH3 motif (19). Key residues in the SH3 domain may play important roles in maintaining proper conformation of beta  proteins.

In summary, we have demonstrated that the signals necessary for membrane targeting of alpha 1C and beta  subunits are likely to be generated by the formation of the complex of alpha 1C and beta  subunits. While the alpha 1C subunit, as well as several mutant and wild-type beta  subunits were unable to target to the plasma membrane when expressed alone, complexes of these subunits were targeted to the plasma membrane, where they exhibited a punctate distribution. In contrast, the alpha 2delta and alpha 1C subunits were unable to achieve this targeting in the absence of the beta  subunits. The results pointed to a key role of beta  subunits in membrane targeting, and the region in the beta  subunit that was responsible for targeting was narrowed to the two domains conserved in all beta  subunits. Moreover, an SH3 motif in the beta  subunits has been suggested to play a role in membrane targeting and association with the alpha 1 subunits. Our studies provided novel biochemical evidence for better understanding the structural determinants of accessory subunit-mediated regulation of L-type calcium channels.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL23306 (to M. M. H.).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.

Dagger Recipient of NIMH, National Institutes of Health, National Research Service Award Predoctoral Fellowship MH10770.

§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., S215, Chicago, IL 60611. Tel.: 312-503-3692; Fax: 312-503-5349; E-mail: mhosey{at}nwu.edu.

The abbreviations used are: DHP, dihydropyridine; BID, beta -interaction domain; SH3, Src homology 3.
    REFERENCES
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Abstract
Introduction
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