Differential Effects of Ca2+ Channel beta 1a and beta 2a Subunits on Complex Formation with alpha 1S and on Current Expression in tsA201 Cells*

Birgit Neuhuber, Uli Gerster, Jörg Mitterdorfer, Hartmut Glossmann, and Bernhard E. FlucherDagger

From the Department of Biochemical Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

To study the interactions of the alpha 1S subunit of the skeletal muscle L-type Ca2+ channel with the skeletal beta 1a and the cardiac beta 2a, these subunits were expressed alone or in combination in tsA201 cells. Immunofluorescence- and green fluorescent protein-labeling showed that, when expressed alone, beta 1a was diffusely distributed throughout the cytoplasm, beta 2a was localized in the plasma membrane, and alpha 1S was concentrated in a tubular/reticular membrane system, presumably the endoplasmic reticulum (ER). Upon coexpression with alpha 1S, beta 1a became colocalized with alpha 1S in the ER. Upon coexpression with beta 2a, alpha 1S redistributed to the plasma membrane, where it aggregated in large clusters. Coexpression of alpha 1S with beta 1a but not with beta 2a increased the frequency at which cells expressed L-type currents. A point mutation (alpha 1S-Y366S) or deletion (alpha 1S-Delta 351-380) in the beta  interaction domain of alpha 1S blocked both translocation of beta 1a to the ER and beta 2a-induced translocation of the alpha 1S mutants to the plasma membrane. However, the point mutation did not interfere with beta 1a-induced current stimulation. Thus, beta 1a and beta 2a are differentially distributed in tsA201 cells and upon coexpression with alpha 1S, form alpha 1S·beta complexes in different cellular compartments. Complex formation but not current stimulation requires the intact beta  interaction domain in the I-II cytoplasmic loop of alpha 1S.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Voltage-activated Ca2+ channels are expressed in a multitude of tissues and are involved in a variety of cell functions (1). The skeletal muscle L-type Ca2+ channel, also called dihydropyridine (DHP)1 receptor, is localized in the junctions between the transverse tubules and the sarcoplasmic reticulum (2, 3), where it primarily functions as the voltage sensor in excitation-contraction coupling (4, 5). It consists of five subunits: the pore-forming alpha 1S subunit, the alpha 2delta subunit complex, a peripheral membrane protein, the beta 1a subunit, and the gamma  subunit, which is unique for this tissue (6-8). Besides functioning as the ion channel proper, the alpha 1S subunit contains the binding sites for specific drugs (9, 10) and domains for interactions with the Ca2+ release channel (11) and the accessory channel subunits (12). The molecular domain responsible for binding the beta  subunit is a conserved motif of nine amino acids in the cytoplasmic loop between repeats I and II of the alpha 1 subunit. Mutations within this motif severely interfered with beta  binding to alpha 1 and with beta -induced current stimulation (13).

The beta  subunit exists in several isoforms and splice variants in different tissues (14). Coexpression of various combinations of alpha 1 subunits and accessory subunits in heterologous expression systems has contributed much of our present knowledge about the functions of the subunits. Whereas the alpha 1 subunit alone is sufficient for the expression of functional Ca2+ channels (15, 16), additional expression of alpha 2delta and beta  subunits increases the incorporation of functional channels into the plasma membrane and modulates the current properties (17-19). beta  coexpression has been demonstrated to enhance the Ca2+ and DHP binding capacity of alpha 1C (20), to elevate current magnitudes, and increase gating charges (21). A multitude of qualitative effects of beta  on Ca2+ currents has been reported. However, they are as variable as the subunit combinations and expression systems used for these studies (14, 22). The most compelling evidence for functional modulation of current properties by beta  are the increase in open probability in single-channel recordings of alpha 1C co-transfected with beta 2a in Xenopus oocytes (23, 24) and in mammalian cells, a modulation of current densities but not of gating charges upon coexpression of alpha 1C with a mutated beta 2a subunit (25). As possible mechanisms for modulatory effects of beta , an improved intramolecular coupling (26) and a stabilization of Ca2+ coordination sites in the channel pore have been suggested (20).

In several recent studies, the mechanism by which beta  increases channel expression in the plasma membrane has been directly addressed. In transiently transfected HEK cells, it was shown with immunocytochemistry that the cardiac beta 2a subunit is preferentially localized in the plasma membrane and that coexpression of the cardiac alpha 1C with beta 2a causes the translocation of alpha 1C into the membrane (25). A similar translocation of a neuronal alpha 1A subunit into the plasma membrane was accomplished by four different beta isoforms: beta 1b, beta 2a, beta 3b, and beta 4 (27). Interestingly, not all of the beta  subunits that are able to induce alpha 1A translocation were localized in the plasma membrane. Only beta 1b and beta 2a but not beta 3b and beta 4 showed a plasma membrane association. Palmitoylation at two N-terminal-located cysteines is a possible mechanism by which beta 2a could be anchored to the plasma membrane. However, mutation of these cysteines that blocked palmitoylation of beta 2a expressed in tsA201 cells caused a failure of current stimulation but not of channel incorporation as seen in recordings of gating charges (28).

The skeletal muscle L-type Ca2+ channel is distinct from other voltage-gated Ca2+ channels with respect to its organization in the triad junction (29), with respect to its function in excitation-contraction coupling (4), and with respect to modulation of its channel properties (14, 30). Whether interactions between the skeletal muscle alpha 1 and beta  subunits expressed in mammalian non-muscle cells also differ from those of other isoforms is not known. Here we report isoform-specific effects of beta 1a and beta 2a on the subcellular distribution and current stimulation upon coexpression with alpha 1S in tsA201 cells. A mutation in the beta  interaction domain of alpha 1S interferes with the stable association of the subunits but not with the beta 1a-induced stimulation of Ca2+ currents.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Lines-- tsA201 cells, a HEK cell subclone stably transfected with the SV40 large T-antigen, were plated and proliferated in F-12 medium (Life Technologies, Inc.) containing 10% fetal bovine serum. Cells were grown to 80% confluency before passing. For structural analysis (green fluorescent protein (GFP) and immunocytochemistry), cells were plated at dilutions of about 1:10 onto poly-L-lysine-coated 13-mm round coverglasses and transfected on the following day (see below) when cells reached 30% confluency. For patch clamp analysis, cells were plated in 35-mm culture dishes at a dilution of 1:10. After transfection at about 50% confluency, cells were replated onto poly-L-lysine-coated 25-mm round coverglasses at dilutions between 1:5 and 1:10 to get isolated cells for patch clamp recordings on the following day.

Transfection-- Transfections were carried out with a liposomal transfection reagent (DOTAP, Boehringer Mannheim) according to the manufacturer's instructions. The total amount of DNA used per 35-mm culture dish was 10 µg, 5-8 µg of which was specific DNA (expression plasmids encoding alpha 1S constructs, beta , and GFP); the rest was filled up with inert DNA (pUC18; Ref. 31). In co-transfection experiments, two or more expression plasmids were combined at equimolar concentrations. This resulted in coexpression of beta GFP with any alpha 1S construct in approximately 70% of beta GFP-transfected cells. The liposome/DNA mixture was diluted in 1.5 ml of F-12 medium and then added to the culture. On the following day, the cells were processed for immunocytochemistry or replated for patch clamp analysis.

Expression Plasmids (see Table I)-- The coding sequence of the rabbit alpha 1S-cDNA was excised from the plasmid pCAC6 (33) by HindIII digestion and inserted into the expression plasmid pcDNA3 (Invitrogen, San Diego, CA).

                              
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Table I
Expression plasmids

Mutations were introduced into alpha 1S by site-directed mutagenesis of the alpha 1S-pcDNA3 using the splicing by overlap extension technique (32). In a first step, a SacII (nt 81 of alpha 1S)-XhoI (nt 2652 of alpha 1S) fragment of the alpha 1S-cDNA was subcloned from the alpha 1S-pcDNA3 into the pBluescript SK(-) (Stratagene, La Jolla, CA). In a second step, the mutations in the alpha 1S were generated by using different mutagenic primers: 1, the sense primer 5'-AAGCAGCAGCTAGAGGAGGACCTTCGGGGCTCCATGAGCTGGAT-3' for a single substitution in the alpha 1S in position 366 from tyrosine to serine (alpha 1S-Y366S); 2, the sense primer 5'-GCCAAGTCCAGGGGAACCTTCCTGAGAGAAGGAAAGCTG-3' for a 30-amino acid deletion mutation in the alpha 1S from position 351 to position 380 (alpha 1S-Delta 351-380); and 3, the sense primer 5'-GCCAAGTCCAGGGGAACCTTCTGGATCACGCAGGGCGAG-3' for an 18-amino acid deletion mutation in the alpha 1S from position 351 to position 368 (alpha 1S-Delta 351-368). To facilitate the identification of positive clones, a PvuII site (nt 1071 of alpha 1S) was eliminated by a silent mutation in the mutagenic primers. The mutated alpha 1S fragments were inserted into the alpha 1S-pcDNA3 after digestion with SacII and XhoI. All mutations were verified by sequence analysis.

The construction of a beta 1a-GFP C-terminal fusion protein required five steps. In the first step the GFP-cDNA was excised from the GFP-pRK5 by using a BamHI (nt 2 of GFP) and a HindIII site (nt 1671 of GFP-pRK5) and ligated into the pBluescript SK(-). Secondly, the SacI (nt 695 of beta 1a)-BamHI (multiple cloning site of pSVL) fragment of the rabbit beta 1a-cDNA from pSVL (20) was inserted into the GFP-pBluescript SK(-). In the third step, a fragment consisting of the 3' terminus of the beta 1a-cDNA fused to the 5' terminus of the GFP-cDNA including a BamHI restriction site was constructed by PCR using the fusion primer: 5'-CTCATGGGATCCATCATGGCGTGCTCCTGCTGTTGGGGCACC-3'. In the fourth step, NarI (nt 1147 of beta 1a) and BamHI (nt 2 of GFP) digestion of the polymerase chain reaction product allowed insertion into the pBluescript SK(-) subclone containing the beta 1a- and GFP-cDNAs. Finally, the fused beta 1a-GFP fragment was inserted into the beta 1a-pcDNA3 after digestion with BstXI (nt 834 of beta 1a) and ApaI (multiple cloning site of pcDNA3). The integrity of the beta 1a-GFP transition was verified by cDNA sequence analysis.

GFP and Immunofluorescence Labeling-- Paraformaldehyde-fixed cultures were immuno-stained as described previously (37). For double-labeling with GFP, Texas red-conjugated antibodies (Jackson Immuno Research, West Grove, PA) were used to exclude bleed-through between the red and the green channels. Working dilutions and the sources of primary antibodies are listed in Table II. Samples were evaluated on a Zeiss Axiovert microscope with epifluorescence and phase contrast optics and documented on 35-mm high speed black and white film. The antibodies were carefully characterized for their use in immunofluorescence experiments in previous studies (37-39). Controls, like the omission of primary antibodies and incubation with inappropriate antibodies, were routinely performed.

                              
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Table II
Antibodies

Patch Clamp Recording-- Electrophysiological recordings were performed using the whole-cell configuration of the patch clamp technique (42). Cultures grown on 25-mm-round coverglasses were mounted in a recording chamber and viewed with a 16× phase contrast multi-immersion lens on a Zeiss Axiovert microscope. Fluorescent cells (beta GFP or GFP-transfected) were selected for recording, except for controls with nontransfected cells. The bath solution contained (40 mM BaCl2, 100 mM tetraethylammonium chloride, 10 mM HEPES, and 5 µM (±)-BayK8644 (adjusted to pH 7.4 using tetrathylammonium hydroxide). Patch pipettes pulled from borosilicate glass and fire-polished were filled with 130 mM cesium aspartate, 10 mM HEPES, 2 mM MgATP, 2 mM CsEGTA, and 0.5 mM MgCl2 (adjusted to pH 7.4 with CsOH). Resistances of the patch pipettes were between 4 and 7 megaohms. An Axopatch 200A patch clamp amplifier controlled by the software pClamp 6.0 (Axon Instruments, Foster City, CA) was used for all recordings. Capacitative currents were compensated using the built-in analog circuits (series resistance error was corrected for 80%). Leak resistance in the cell-attached mode was normally larger (8 gigaohms). Current data were low-pass Bessel-filtered with 1 kHz and sampled at 0.5 kHz with an IBM-compatible PC. To test cells for the expression of barium currents, a voltage ramp protocol was used (from -80 mV to +70 mV over a period of 750 ms) without using any leak current subtraction. Under these conditions, the current detection threshold, indicated by an inward peak in the current trace (see, e.g. Fig. 6a), was about 10 pA. Nontransfected tsA201 cells or cells transfected only with beta GFP showed one or, in rare cases, two distinct types of inward barium currents. One with the current peak at -11.5 mV ± 2.1 mV S.D. during the voltage ramp and with fast inactivation kinetics characteristic for T-type current; a second current peaking at 8.7 mV ± 5.1 mV S.D. (see also Ref. 43). To exclude a possible interference with the analysis of the heterologously expressed currents, any current peaking below +30 mV was rejected from the analysis. Only cells transfected with the alpha 1S constructs showed inward barium currents, with the current peak located between +30 mV and + 50 mV of the voltage ramp. These high voltage-activated currents were therefore attributed to the expression of one of the alpha 1S constructs. For a further check, I/V curves were determined for those cells showing stable recordings with a high current density using a voltage step protocol with a holding potential of -80 mV and 1 s test pulses of -40 mV in 10 mV increments to +80 mV (using a -P/4 leak subtraction protocol). The I/V curves and current kinetics (activation time constant tau act = 18.2 ms ± 3.5 ms S.E., inactivation time constant tau act = 486 ms ± 41 ms S.E. at +40 mV test potential and alpha 1S + beta GFP coexpression; see Fig. 6b) showed similar characteristics when alpha 1S and beta 1a were coexpressed in L-cells (18) or in myotubes (44).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

beta 1a and alpha 1S Are Expressed in Different Cellular Compartments-- An expression plasmid encoding a fusion protein of the beta 1a subunit of the skeletal muscle DHP receptor and GFP, called beta GFP, has been constructed as a tool for studying the subcellular distribution of beta 1a transiently expressed in tsA201 cells. beta GFP aids in the identification of cells expressing the beta 1a subunit and allows its direct localization. Double-labeling of transfected tsA201 cells with GFP and immunofluorescence using an antibody against the beta  subunit resulted in identical distribution patterns of both stains (Fig. 1, a and b). Transfection with the beta GFP fusion protein (Fig. 1b) or transfection with the wild type beta 1a (Fig. 1e) also resulted in identical beta  immuno-labeling patterns. Thus, beta GFP fluorescence gives a true picture of the beta 1a localization, and using the fusion protein instead of the wild type beta 1a, has no undesired effects on its subcellular localization. This was still true when the beta  distribution was altered upon coexpression with the alpha 1S subunit (see below). Moreover, electrophysiological analysis showed that effects of beta GFP and wild type beta 1a on Ca2+ currents were indistinguishable in control experiments in which the cardiac alpha 1C was coexpressed with beta GFP or beta 1a2 and upon coexpression of both beta  constructs with alpha 1S in dysgenic skeletal myotubes (53). Thus, our beta GFP construct can be used in place of beta 1a, with the advantage of allowing the direct observation of beta 1a expression and localization in transiently transfected cells. The distribution pattern of beta GFP when expressed by itself in tsA201 cells was always diffuse and evenly distributed throughout the cytoplasm (Fig. 1a). The label did not appear to be specifically associated with any membrane system or cytoplasmic structure. Only the nuclei and vacuolar structures excluded the beta GFP stain. This was different from the fluorescence signal obtained by expressing GFP alone, which was also diffusely distributed throughout the cytoplasm but in addition labeled the nuclei (Fig. 1d).


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Fig. 1.   Subcellular localization of the DHP receptor beta 1a subunit expressed in tsA201 cells. A fusion protein of beta 1a and GFP (beta GFP) is expressed in a tsA201 cell (a-c) and localized with GFP fluorescence (a) and by immunofluorescence with a beta  antibody (b). Both localization methods give a very similar diffuse distribution pattern of beta GFP. Cells transfected with GFP alone also show a diffuse fluorescence, but unlike with beta GFP, the nuclei are brightly labeled (d). tsA201 cells transfected with the wild type beta 1a and immuno-labeled with anti-beta (e) show the same diffuse distribution as beta GFP (cf. b). e and f, phase contrast (Ph) images of cells shown in a, b and in e, respectively. N, nuclei of transfected cells; bar, 10 µm.

The alpha 1S subunit of the DHP receptor expressed in tsA201 cells showed a different subcellular distribution. Localizing alpha 1S with a specific antibody showed that it was contained in a dense network of a tubular membrane compartment that extended throughout the entire cytoplasm (Fig. 2, a, c, and e). The tubular/reticular network was very dense in the perinuclear region, and occasionally the nuclear envelope was also labeled. The reticular nature of the compartment could be best seen in thin regions of the cells. Based on these structural characteristics, the alpha 1S-containing compartment is identified as the ER. Other cytoplasmic compartments that could have been recognized by their morphology, most importantly, the plasma membrane, were not labeled with the alpha 1S antibody. Transfection of tsA201 cells with alpha 1S constructs in which the beta  interaction domain has either been deleted or mutated showed the same subcellular distribution in the ER (see Fig. 2, a, c, and e).


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Fig. 2.   Subcellular localization of the wild type and two mutants of the DHP receptor alpha 1S subunit. tsA201 cells transfected with alpha 1S (a and b), alpha 1S-Y366S (c and d), and alpha 1S-Delta 351-380 (e and f) were immunofluorescence-labeled with an antibody against the alpha 1S subunit. All three alpha 1S constructs are expressed in a cytoplasmic membrane system that is densest in the perinuclear region of the cells (saturated fluorescence signal around the nuclei (N)). Focusing on thin regions of the cells reveals the reticular nature of the alpha 1-containing membrane system. a, c, and e are enlargements of the regions indicated by boxes in the corresponding phase contrast (Ph) images (b, d, and f). Bars, 10 µm.

Coexpression of beta 1a and alpha 1S Causes the Redistribution of beta 1a but Not of alpha 1S-- beta GFP expressed alone was diffusely distributed throughout the cytoplasm (Fig. 3, a and b). However, when beta GFP was coexpressed with the wild type alpha 1S subunit of the DHP receptor, the beta 1a subunit became localized in the tubular/reticular membrane system, presumably the ER (Fig. 3, c and d). The cytoplasm was essentially free of diffuse beta GFP fluorescence, and no staining of the plasma membrane could be observed. Double-labeling of beta GFP and an antibody against alpha 1S showed a colocalization of beta 1a and alpha 1S in the ER (see Fig. 4, a and b). To test whether this redistribution of beta GFP was due to its direct association with the alpha 1S subunit, three alpha 1S constructs with mutations in the beta  interaction domain have been generated. In alpha 1S-Delta 351-380, the entire beta  interaction domain has been deleted; in alpha 1S-Delta 351-368, the N-terminal portion of the beta  interaction domain has been deleted; in alpha 1S-Y366S, tyrosine in position 366 has been substituted with serine. Equivalent mutations in the beta  interaction domain of alpha 1A have been shown to inhibit binding of beta 1b and current stimulation (12). If the translocation of beta GFP from the cytoplasm to the ER was a result of a direct association of beta 1a to alpha 1S, mutations in the beta  interaction domain of alpha 1S should inhibit the observed translocation of beta GFP.


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Fig. 3.   Subcellular distribution of beta 1a coexpressed with the wild type and mutant alpha 1S subunits. tsA201 cells were transfected with beta GFP alone (a and b) or in combination with alpha 1S (c and d), alpha 1S-Y366S (e and f), and alpha 1S-Delta 351-380 (g and h). beta 1a is localized by GFP fluorescence (upper row) and with immunofluorescence using an antibody against the beta  subunit (lower row). When coexpressed with the wild type alpha 1S, the beta 1a subunit is localized in a reticular cytoplasmic membrane system (arrow; c and d). Upon coexpression with alpha 1S constructs mutated in the beta  interaction domain, beta 1a remains diffusely distributed throughout the cytoplasm (e-h), similar to the distribution pattern in cells in which beta GFP has been expressed alone (a and b). N, nuclei; bar, 20 µm.


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Fig. 4.   Comparison of the subcellular localization of beta 1a and alpha 1S in tsA201 cells coexpressing beta GFP with the wild type and mutant alpha 1S subunits. beta 1a localization is shown with GFP fluorescence (upper row), and alpha 1S localization is shown with immunofluorescence (lower row). beta GFP and wild type alpha 1S are colocalized in a reticular cytoplasmic membrane system (a and b). alpha 1S-Y366S (d) and alpha 1S-Delta 351-380 (f) are both localized in the reticular membrane system; however, in these cells beta GFP is not associated with the reticular membrane system but diffusely distributed in the cytoplasm (c and e). Insets show a small field of a thin area of the cell at 2-fold magnification. N, nuclei; bar, 10 µm.

Indeed, when beta GFP was coexpressed with the alpha 1S constructs in which the beta  interaction domain had been mutated, the beta GFP distribution was diffuse (Fig. 3, e-h) and indistinguishable from that of beta GFP expressed alone (Fig. 3, a and b). Quantitative analysis of the beta GFP distribution when expressed alone or in combination with either one of the alpha 1S mutants showed that 376 of 441 (85.3%) analyzed cells co-transfected with alpha 1S had a clearly identifiable ER distribution of beta GFP, but in none of the cells co-transfected with alpha 1S-Y366S or alpha 1S-Delta 351-380 was beta GFP found in the ER (Table III). The small fraction of cells in beta GFP/alpha 1S co-transfected cultures that did not show an ER distribution of beta GFP most likely represents cells that only expressed beta GFP and not alpha 1S and cells with an overall poor morphology. The differential distribution of beta GFP upon coexpression with the different alpha 1S constructs was verified by immunolocalization of beta 1a (Fig. 3, b, d, f, and h). Immuno-labeling of beta 1a or beta GFP gave the same distribution patterns as seen with intrinsic beta GFP fluorescence (Fig. 3, a, c, e, and g). 348 of 447 (77.9%) analyzed cells co-transfected with beta GFP and alpha 1S, but none of the cells co-transfected with either one of the alpha 1S mutants showed beta  immuno-localized in the ER. Since the two deletion mutants, alpha 1S-Delta 351-368 and alpha 1S-Delta 351-380, gave identical results in all experiments, quantitative data and figures of only alpha 1S-Delta 351-380 will be shown in this article as representative results for both constructs. Coexpression of the alpha 1S constructs with the wild type beta 1a gave the same results in that only cells co-transfected with beta 1a and the wild type alpha 1S showed an ER distribution pattern of beta  immunostain (see Table III). Thus, the coexpression of beta GFP or beta 1a with the wild type alpha 1S caused the redistribution of the beta 1a subunit from the cytoplasm to a tubular/reticular membrane compartment, presumably the ER, but this redistribution fails when the beta  interaction domain in the alpha 1S subunit is mutated.

                              
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Table III
Subcellular distribution of alpha 1S, beta 1a, and beta GFP in transfected tsA201 cells
Values are given in cell numbers and percentage for each individual construct; balance to 100% represents cells with labeling patterns that could not unambiguously be identified as ER or cytoplasmic (Cyt.) stain.

Double-labeling of the beta 1a subunit and the alpha 1S constructs with GFP and a specific antibody, respectively, showed that upon coexpression of beta GFP and the wild type alpha 1S, beta GFP was colocalized with the alpha 1S subunit (Fig. 4, a and b). The ER labeling patterns of alpha 1S and beta GFP were identical. However, when beta GFP was expressed together with alpha 1S-Y366S or alpha 1S-Delta 351-380, beta GFP remained diffuse and was not colocalized with the tubular/reticular compartment containing the mutant alpha 1S subunits (Fig. 4, c-f). In contrast, no changes in the distribution of the wild type or mutant alpha 1S constructs occurred upon coexpression with beta GFP. This indicates that specific interactions between the beta  interaction domain of alpha 1S and beta 1a cause the association of beta GFP with the alpha 1S subunit at the ER. However, coexpression of alpha 1S and beta 1a does not result in a recognizable change of alpha 1S distribution within the cell.

Coexpression of beta 2a and alpha 1S Causes the Redistribution of alpha 1S but Not of beta 2a-- Expression of the cardiac isoform of the DHP receptor beta  subunit (beta 2a) alone or in combination with the skeletal muscle alpha 1S subunit gave a different distribution pattern than that seen with beta 1a. tsA201 cells transfected with beta 2a and immuno-stained with the beta  antibody showed an unequivocal plasma membrane stain (Fig. 5a). The periphery of the cells was outlined by a continuous fine line, and the cytoplasm was essentially free of immuno-label. The plasma membrane stain was more clearly seen in regions where the plane of the membrane was parallel to the optical axes rather than in flat parts of the cells, where the membrane was almost perpendicular to the optical axes. This plasma membrane label of beta 2a did not change when it was coexpressed with any of our alpha 1S subunit constructs (Fig. 5, c, e, and g). However, the distribution pattern of wild type alpha 1S changed upon coexpression with beta 2a. Whereas the ER and nuclear envelope label could still be seen in some cells, most tsA201 cells coexpressing beta 2a and alpha 1S showed a distribution of alpha 1S in the periphery of the cells (Fig. 5d). 279 of 395 (70.6%) cells co-transfected with beta 2a and alpha 1S had a clearly identifiable peripheral localization of alpha 1S (Table IV). The remaining cells, which for the most part had alpha 1S localized in the ER, could often be identified as those that expressed alpha 1S without beta 2a. The peripheral alpha 1S stain was colocalized with the beta 2a immuno stain in the plasma membrane. However, the alpha 1S label was not continuous throughout the plasma membrane but occurred in intensively labeled aggregates in or near the plasma membrane. These aggregates were of variable sizes and shapes. The alpha 1S distribution was always distinct from that of the beta 2a. Whereas beta 2a was sometimes more intensive in the alpha 1S aggregates, more often it was not, and it always continued between the alpha 1S aggregates. Thus, alpha 1S is colocalized with beta 2a, but they do not coexist throughout the plane of the membrane.


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Fig. 5.   Comparison of the subcellular localization of beta 2a and alpha 1S in tsA201 cells coexpressed with the wild type and mutant alpha 1S subunits. tsA201 cells were transfected with the cardiac beta 2a alone (a and b) or in combination with alpha 1S (c and d), alpha 1S-Y366S (e and f), and alpha 1S-Delta 351-380 (g and h) and were double-immunofluorescence-labeled. beta 2a (upper row) is localized in the periphery of the cell (arrows). This label is best seen in areas where the plasma membrane is oriented parallel to the optical axes. Wild type alpha 1S is localized in discrete aggregates in the cell periphery (d, examples indicated by open arrowheads) and in the nuclear envelope (filled arrowheads). alpha 1S-Y366S (f) and alpha 1S-Delta 351-380 (h) do not form these peripheral clusters but remain localized in a tubular/reticular membrane system, presumably the ER (double arrowheads). N, nuclei; bar, 10 µm.

                              
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Table IV
Subcellular distribution of alpha 1S, and beta 2a in transfected tsA201 cells
Values are given in cell numbers and percentage for each individual construct; balance to 100% represents cells with labeling patterns that could not unambiguously be identified as plasma membrane (PM) or ER stain.

This translocation of alpha 1S from the ER to the plasma membrane upon coexpression with beta 2a was only seen with the wild type alpha 1S. Coexpression of beta 2a with either one of the mutant alpha 1S constructs had no effect on the distribution of the alpha 1S constructs (Table IV). alpha 1S-Y366S and alpha 1S-Delta 351-380 remained localized in the ER (Fig. 5, f and h). This indicates that a specific interaction between alpha 1S and beta 2a is involved in the alpha 1S translocation and that this interaction requires the intact beta  interaction domain in alpha 1S.

Coexpression of beta 1a but Not of beta 2a Enhances the Frequency of Ca2+ Current Expression-- The rate at which tsA201 cells expressed Ca2+ current after transfection with alpha 1S alone or in combination with beta 1a or beta 2a was used to assess the capability of beta  subunits to facilitate the insertion of functional Ca2+ channels in the plasma membrane. Cells positively identified as transfected by expression of GFP fluorescence were tested for expression of Ca2+ currents with the whole-cell patch clamp method using a voltage ramp protocol. High voltage-activated Ca2+ currents were identified by an inward current peak at potentials above +30 mV during the ramp depolarization (see "Experimental Procedures" and Fig. 6a). Such Ca2+ currents were found in 14 of 40 (35%) tested cells co-transfected with alpha 1S and beta GFP and in 10 of 33 (30%) cells co-transfected with alpha 1S-Y366S and beta GFP but in none of 25 cells co-transfected with alpha 1S-Delta 351-380 and beta GFP. When the alpha 1S constructs were expressed without the beta 1a subunit, the expression frequency of high voltage-activated currents was close to zero; alpha 1S, 1 of 38 (2.6%) and alpha 1S-Y366S, 0 of 46 (0%; Fig. 6a). The increased frequency of Ca2+ current expression upon coexpression of beta GFP suggests that the beta 1a subunit enhances current expression by alpha 1S. Interestingly, this effect of beta GFP was not limited to the wild type alpha 1S, the only alpha 1S construct that showed a structural association with the beta 1a subunit, but was also observed with alpha 1S-Y366S. Thus, coexpression of the skeletal muscle beta 1a subunit significantly increased current expression by the wild type alpha 1S and the alpha 1S-Y366S (p < 0.001), whereas alpha 1S-Delta 351-380 did not support expression of high voltage-activated current expression, even in the presence of the beta 1a subunit. Due to the rare occurrence of current expression in cells expressing alpha 1S without beta 1a, we did not attempt further analysis of beta 1a effects on current properties. The few cells analyzed with a voltage-step protocol do not suggest differences in voltage dependence, current kinetics, or current densities between cells expressing alpha 1S with and without beta 1a.


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Fig. 6.   Expression frequency of high-voltage-activated Ca2+ currents in tsA201 cells co-transfected with alpha 1S or alpha 1S-Y366S with and without beta 1a (beta GFP) or beta 2a (plus GFP). a, expression of a high voltage-activated Ca2+ current was identified by an inward current peak above +30 mV during a voltage ramp depolarization from -80 to + 70 mV (right panel; see "Experimental Procedures"). Numbers give the fraction of positive cells of the total number of cells tested. Co-transfection with beta GFP caused a significant increase in current expression compared with the poor expression without a beta  subunit (p < 0.001, left panel). The difference in current expression between alpha 1SY366S and alpha 1SY366S plus beta 2a is not significant (p > 0.05). b, representative current traces during voltage steps from a holding potential of -80 mV to the indicated test potentials (left panel) and the corresponding peak current/voltage relationship (right panel). Data shown in a, right panel, and b are from the same cell co-transfected with a1S and beta GFP. pA/pF, picoamperes/picofarads.

Since coexpression of the cardiac beta 2a subunit caused a marked translocation of the skeletal muscle alpha 1S subunit to the plasma membrane (see above), we wanted to know how this affected current expression in tsA201 cells. However, contrary to our expectations, expression of Ca2+ currents was as poor and not significantly different from that when alpha 1S or alpha 1S-Y366S was expressed without a beta  subunit (Fig. 6a). Currents were only found in 1 of 51 (2%) cells transfected with beta 2a plus the wild type alpha 1S and in 4 of 43 (9%) cells transfected with beta 2a plus alpha 1S-Y366S. Thus, beta 2a lacks the ability to increase the frequency at which skeletal Ca2+ currents are expressed in tsA201 cells. Apparently, the function of beta 2a responsible for the translocation of alpha 1S to the plasma membrane (see above) is not coupled to beta -induced stimulation of Ca2+ current expression.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present study we used heterologous expression of the alpha 1S subunit of the skeletal muscle L-type Ca2+ channel with and without the skeletal and cardiac beta  subunit isoforms to investigate the mechanisms and functions of their interactions. A fusion protein of beta 1a and the green fluorescent protein and alpha 1S constructs with modifications in the known beta  interaction domain in the cytoplasmic loop between repeats I and II of alpha 1S have been generated. The effects of coexpressing these constructs on the subcellular distribution of the channel subunits were analyzed with GFP- and immunofluorescence-labeling, and the effects on expression of Ca2+ currents were tested with patch clamp recordings. The results indicate that two beta  subunit isoforms, beta 1a and beta 2a, that differ in their intrinsic ability to associate with the plasma membrane also differ in their effects on the subcellular distribution of the alpha 1S·beta complex and on the expression of functional channels.

Subcellular Distribution of beta 1a and beta 2a-- The beta 1a subunit of the skeletal muscle DHP-receptor or its GFP fusion protein (beta GFP), expressed alone in tsA201 cells, was localized diffusely throughout the cell. The labeling pattern was similar to that of GFP alone, and no preferential association with the plasma membrane or any other cellular structure was seen. Thus, the hydrophilic beta 1a subunit appears to lack a specific anchoring mechanism for the plasma membrane. This localization was different from that of beta 2a, which was preferentially localized in the plasma membrane when expressed alone in tsA201 cells. An association with the plasma membrane has previously been shown for beta 1b and beta 2a when expressed without a alpha 1 subunit in heterologous mammalian expression systems (25, 27), and palmitoylation of two N-terminal cysteines was suggested as possible anchoring mechanism of beta 2a (28). Other beta  subunit isoforms, such as beta 3b and beta 4, were not localized in the plasma membrane but were distributed throughout the cell when expressed in COS-7 cells (27). Thus, beta 1a shares the diffuse distribution with beta 3b and beta 4. Even though the distribution pattern of beta 1a strongly suggests a cytoplasmic localization, it does not rule out the possibility that beta 1a is, at least in part, associated with some diffusely distributed membrane or cytoskeletal components. Significant amounts of beta 1a were found in the particulate fraction as well as in the supernatant when cells were fractionated by centrifugation at 70,000 × g (not shown). This is consistent with published data showing other beta  isoforms, including the non-plasma membrane-associated beta 3b and beta 4, in the particulate fraction (27) and with data showing that the removal of the membrane anchor of beta 2a did not change its distribution between supernatant and particulate fraction (28). However, the redistribution of beta 1a from a cytoplasmic localization to the ER upon coexpression with alpha 1S suggests that beta 1a is not tightly bound to other cytoplasmic structures but is readily available for association with the alpha 1S subunit.

Association and Functional Expression of beta 1a and alpha 1S-- How do beta 1a and beta 2a, which by themselves are localized in distinct cytoplasmic compartments, interact with the alpha 1S subunit, and how does this interaction affect localization and expression of functional channels in the membrane? The wild type and the mutant alpha 1S subunits of the skeletal muscle DHP receptor were all localized in a tubular/reticular cytoplasmic membrane system, most likely the ER. No plasma membrane association of alpha 1S was observed with immunocytochemistry, and essentially no skeletal-type Ca2+ currents were recorded in tsA201 cells transfected with any of the alpha 1S constructs alone. The latter is concordant with the generally observed poor current expression with the skeletal muscle alpha 1S isoform in heterologous expression systems as compared with other alpha 1 subunit isoforms. Thus, without a beta  subunit, most of the expressed alpha 1S was retained in the ER, whereas only little or none was inserted into the plasma membrane.

When beta 1a was coexpressed with the alpha 1S subunit, the subcellular distribution of beta 1a changed dramatically, whereas the localization of alpha 1S appeared unaltered. beta 1a was now colocalized with the alpha 1S subunit in the ER. The redistribution of beta 1a upon coexpression with alpha 1S was blocked when the beta  interaction domain in alpha 1S had been mutated (alpha 1S-Y366S) or deleted (alpha 1S-Delta 351-380). This indicates that the alpha 1S subunit is sufficient for the association of beta 1a with the DHP receptor complex and that this interaction depends on the intact beta  interaction domain that has been identified in the cytoplasmic loop between repeats I and II of all examined alpha 1 subunits (12, 13). In a parallel study in dysgenic myotubes, we found that this stable alpha 1S-beta 1a association is required for the normal targeting of the beta 1a subunit but not of the alpha 1S into the skeletal muscle triad (53). Thus, this interaction is of importance in the native system. The fact that alpha 1S and beta 1a associate with one another at the ER shows that neither the processing of the alpha 1S subunit in the biosynthetic pathway nor the transport of alpha 1S to the plasma membrane are necessary before the formation of the alpha 1S·beta 1a complex. Thus it is possible that in the native system the association of alpha 1S and beta 1a also occurs in the ER, which would be a prerequisite for a chaperon-like function of the beta subunit in the transport of alpha 1S to the plasma membrane (27).

Indeed, upon coexpression of alpha 1S and beta 1a, Ca2+ currents could be recorded, indicating that beta 1a is required for the expression of functional channels in the plasma membrane of transfected tsA201 cells. However, current expression was not accompanied by detectable plasma membrane staining of either alpha 1S or beta 1a. Therefore, measurable Ca2+ currents can be supported by a concentration of channels in the plasma membrane that is too low to be detected with immunocytochemistry. The highest current densities recorded in our experiments correspond to approximately 10 active channels/µm2. Even if this number represents an underestimate due to the possible presence of silent channels, the expected density in the plasma membrane of the tsA201 cells would be far below the density of approximately 2,200 channels/µm2 found in skeletal muscle triads where this channel can be reliably localized with the same antibody. Despite the fact that patch clamp analysis gives evidence of channels in the plasma membrane that cannot be visualized by immunofluorescence labeling, one can assume that these channels show the same behavior with respect to association with beta  as the channels observed in the ER, since both originate from the same expression plasmids. This is supported by the finding that deletion of the beta  interaction domain resulted in failure of both alpha 1S·beta 1a complex formation and current expression. Most interesting and unexpected, however, was the finding that current expression was still induced when beta 1a was coexpressed with the Y366S mutant alpha 1S, which did not stably associate with beta 1a. This differential behavior of alpha 1S-Y366S with respect to stable association with beta 1a and current stimulation by beta 1a indicates that the two properties of beta 1a are independent of each other and that alpha 1S·beta 1a complex formation but not necessarily current stimulation utilizes the known beta  interaction domain in the I-II cytoplasmic loop of alpha 1S.

Translocation of alpha 1S upon Coexpression with beta 2a-- beta 2a expressed alone was localized in the plasma membrane of tsA201 cells. Its localization did not change upon coexpression with alpha 1S, but beta 2a coexpression induced the translocation of the alpha 1S subunit from the ER to the plasma membrane. The beta 2a-induced translocation of alpha 1S was only observed with the wild type alpha 1S but not with mutants in which the beta  interaction domain had been altered or deleted. This indicates that a specific interaction of the membrane-bound beta 2a with the beta  interaction domain in the I-II cytoplasmic loop of alpha 1S is involved in incorporating the alpha 1 subunit in the plasma membrane. However, alpha 1S and beta 2a were not precisely colocalized in the plasma membrane. Although beta 2a remained evenly distributed throughout the membrane, alpha 1S was localized in aggregates of variable sizes and shapes. Apparently, beta 2a facilitates the incorporation of the alpha 1S subunit into the plasma membrane but does not remain tightly associated with alpha 1S when this subunit forms aggregates. Even though beta 2a did not modulate alpha 1S function, the ability of beta 2a to reversibly bind to alpha 1 subunits may be related to its mechanism of modulation of other alpha 1 subunit isoforms. For instance, it has been suggested that beta  competes with G-protein beta gamma subunits for the binding site in the I-II cytoplasmic loop of neuronal Ca2+ channel isoforms (45-47). For such a competition to occur, free beta  and G-protein beta gamma subunits must be available. A beta  subunit anchored in the plasma membrane like the G-protein beta gamma subunits may have an advantage in such a competitive interaction over beta  subunits distributed in the cytosol. Our observation that beta 2a is uniformly distributed in the plasma membrane and not necessarily concentrated in the alpha 1S-containing membrane patches is consistent with the existence of free beta  in the plasma membrane of alpha 1S-expressing cells.

A beta -dependent translocation of the cardiac alpha 1C and the neuronal alpha 1A subunits by membrane-bound beta 1b and beta 2a has been reported (25, 27, 28). However, coexpression of these subunit isoforms also resulted in an increase of current densities compared with cells expressing alpha 1 subunits alone. This differs from the effects of beta 2a on the skeletal muscle alpha 1S subunit that did not lead to an induction of current expression. The structural basis of such isoform-specific properties of a beta  subunit may be the modulatory binding site in the N terminus of the beta  subunit (48, 49). Nevertheless, the cardiac beta 2a isoform promoted the plasma membrane incorporation of alpha 1S. Thus, beta  isoforms differ in their ability to target the alpha 1 subunit to the plasma membrane as well as in their ability to functionally modulate the alpha 1 subunit. Surprisingly, the targeting function and induction of current expression are not necessarily linked. alpha 1S current stimulation may be a specific property of the beta 1 isoforms, and plasma membrane localization of a beta  subunit may be important for quantitative incorporation of alpha 1S into the plasma membrane. Then the combination of the two properties, as seems to be the case for beta 1b, might be the key for efficient expression of the skeletal muscle Ca2+ channel in heterologous expression systems (30). This idea is consistent with the findings of a recent study showing that expression of skeletal Ca2+ currents in Xenopus oocytes is dramatically enhanced by coexpression of alpha 1S with beta 1b but not with beta 1a (50).

Differential alpha 1-beta Interactions-- Our present results demonstrate that alpha 1-beta interactions affect both membrane targeting and the induction of currents in an isoform-specific manner. The structural changes induced by beta 1a and beta 2a, even though opposite in direction, were both dependent on the intact beta  interaction domain in the cytoplasmic loop between repeats I and II of the alpha 1S subunit. In one case, translocation of beta 1a to the ER failed; in the case of beta 2a, translocation of alpha 1S to the plasma membrane failed when the beta  interaction domain was mutated or deleted. Thus the beta  interaction domain in the I-II cytoplasmic loop of alpha 1S is critical for complex formation with different beta  subunit isoforms. Whether the beta  isoform is membrane-anchored or not determines where in the cell the association occurs. In contrast, the ability of the beta 1a subunit to induce expression of Ca2+ currents is isoform-specific (beta 2a failed to stimulate currents), and neither requires the intact beta  interaction domain nor the stable association of alpha 1S and beta 1a.

These differential effects of beta 1a and beta 2a on the wild type and mutant alpha 1S subunits can be explained by two different models of alpha 1S-beta interactions. The first model assumes that the beta  interaction domain in the I-II cytoplasmic loop of alpha 1S mediates all observed effects of beta . This is supported by our observation that deletion of the beta  interaction domain blocks complex formation with either beta  isoform as well as current stimulation upon coexpression of beta 1a. The tyrosine-to-serin substitution of one of the nine conserved residues in the beta  interaction domain may according to this model lower the affinity enough to perturb complex formation without impeding the interaction that is required for the expression of functional channels in the membrane. However, alpha 1S membrane targeting by beta 2a, which also required interactions with the intact beta  interaction domain, did not stimulate Ca2+ currents. This shows that the association of a beta  subunit with alpha 1S via the conserved beta  interaction domain in the I-II cytoplasmic loop does not lead to induction of Ca2+ currents. Therefore, interactions with the beta  interaction domain in the I-II cytoplasmic loop are necessary but not sufficient for beta -induced current stimulation. An alternative model, assuming the existence of a second domain in alpha 1S for isoform-specific functional interactions with beta , could explain these observations. Whereas the primary beta  interaction domain in the I-II cytoplasmic loop of alpha 1S binds all known beta  subunits with high affinity (13, 51), a second beta  interaction domain may specifically interact with certain beta  isoforms with a low affinity that by itself does not permit the stable association of the two subunits. In the native system, binding via the primary interaction domain may direct beta 1a into position to functionally interact with the secondary binding site. In the heterologous expression system, high concentrations of beta 1a in the cytoplasm may allow this functional interaction even without the tight association of the two subunits. This model is supported by recent evidence for the existence of a second binding site for the beta  subunits in the C terminus of the alpha 1E subunit (52) as well as by evidence for isoform-specific domains in the beta  subunit that are involved in functional modulation of alpha 1 subunits (48, 49).

In summary, the experiments reported here allow the distinction between functional interactions of the Ca2+ channel alpha 1S and beta  subunits and their complex formation. Differences in the subcellular distribution of beta  subunits and the occurrence of common as well as isoform-specific effects on the skeletal muscle alpha 1S subunit are indicative of the complex regulation of Ca2+ channels by beta  subunits. Whether the distinct types of alpha 1S-beta 1a interactions are mediated by one or more interaction domains of alpha 1S remains to be demonstrated.

    ACKNOWLEDGEMENTS

We thank Drs. S. Froehner and J. Striessnig for their generous gift of antibodies, Drs. T. Kamp, Y. Seino, and F. Döring for their generous gift of expression plasmids, and Dr. G. Eaholtz for the generous gift of tsA201 cells. We also thank Dr. H. Hoflacher for excellent technical assistance.

    FOOTNOTES

* This work was supported in part by the Fonds zur Förderung der wissenschaftlichen Forschung, Austria, Grants S06612-MED (to B. E. F.) and S06601-MED (to H. G.), the Austrian National Bank Grant 3535, and the European Commissions Training and Mobility of Researchers Network Grant ERBFMRXCT 960032 (to B. E. F.).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 An Austrian Program for Advanced Research and Technology (APART) fellow of the Austrian Academy of Sciences. To whom correspondence should be addressed: Dept. of Biochemical Pharmacology, University of Innsbruck, Peter-Mayr-Str. 1, A-6020 Innsbruck, Austria. Tel.: 43-512-507-3167; Fax: 43-512-507-2858; E-mail: bernhard.e.flucher{at}uibk.ac.at.

1 The abbreviations used are: DHP, dihydropyridine; GFP, green fluorescent protein; beta GFP, fusion protein of beta 1a and GFP; nt, nucleotide; ER, endoplasmic reticulum.

2 B. Neuhuber, U. Gerster, and B. E. Flucher, unpublished results.

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Abstract
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Results
Discussion
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