The α1-β-Subunit Interaction That Modulates Calcium Channel Activity Is Reversible and Requires a Competent α-Interaction Domain

Abstract

High voltage-gated calcium channels consist of a pore-forming subunit (α1) and three nonhomologous subunits (α2/δ, β, and γ). Although it is well established that the β-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the α1-subunit remains controversial. Here, we address this issue by examining the effect of purified β2a protein on CaV1.2 and CaV2.3 channels expressed in Xenopus oocytes. The β2a-subunit binds to the α1-interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of CaV1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the α1-β interaction through the AID site can take place at the plasma membrane. Furthermore, injection of β2a protein inhibited inactivation of CaV2.3 channels and converted fast inactivating CaV2.3/β1b channels to slow inactivating channels. Inhibition of inactivation required larger concentration of β2a in oocytes expressing CaV2.3/β1b channels than expressing CaV2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the α1-β interaction is reversible in intact cells and defines calcium channels β-subunits as regulatory proteins rather than stoichiometric subunits. High voltage-gated calcium channels consist of a pore-forming subunit (α1) and three nonhomologous subunits (α2/δ, β, and γ). Although it is well established that the β-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the α1-subunit remains controversial. Here, we address this issue by examining the effect of purified β2a protein on CaV1.2 and CaV2.3 channels expressed in Xenopus oocytes. The β2a-subunit binds to the α1-interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of CaV1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the α1-β interaction through the AID site can take place at the plasma membrane. Furthermore, injection of β2a protein inhibited inactivation of CaV2.3 channels and converted fast inactivating CaV2.3/β1b channels to slow inactivating channels. Inhibition of inactivation required larger concentration of β2a in oocytes expressing CaV2.3/β1b channels than expressing CaV2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the α1-β interaction is reversible in intact cells and defines calcium channels β-subunits as regulatory proteins rather than stoichiometric subunits. High voltage-gated calcium channels are multi-subunit proteins complexes where a pore-forming subunit combines with one or more nonhomologous auxiliary subunits (1Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1952) Google Scholar). One of these auxiliary subunit, the β-subunit, is crucial for channel function, because in addition to stimulating channel activity it appears to be required for surface expression of the channel protein (2Tareilus E. Roux M. Qin N. Olcese R. Zhou J.M. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1703-1708Crossref PubMed Scopus (154) Google Scholar). These two effects combined result in a severalfold increase in the ionic current density in heterologous expression systems, but the relative contribution, biological relevance, and extent to which both processes are independent from each other remain elusive. Early studies show that in Xenopus oocytes, coexpression of β2a with the pore-forming α1 subunit from cardiac cells (CaV1.2) augments ionic currents mostly by increasing ionic current to charge movement ratio (3Neely A. Wei X. Olcese R. Birnbaumer L. Stefani E. Science. 1993; 262: 575-578Crossref PubMed Scopus (203) Google Scholar). Later on, it was shown that the addition of the β-subunit as purified protein is capable of modulating channel activity of the α1 subunit expressed in Xenopus oocytes (4Neely A. Garcia-Olivares J. Voswinkel S. Horstkott H. Hidalgo P. J. Biol. Chem. 2004; 279: 21689-21694Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 5Yamaguchi H. Hara M. Strobeck M. Fukasawa K. Schwartz A. Varadi G. J. Biol. Chem. 1998; 273: 19348-19356Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and also on isolated membranes from skeletal muscle (6Garcia R. Carrillo E. Rebolledo S. Garcia M.C. Sanchez J.A. J. Physiol. 2002; 545: 407-419Crossref PubMed Scopus (23) Google Scholar). These results suggest that modulation of function is separated from the effect on channel expression and predicts that binding sites remain available on the mature channel. However, the α1-β-subunit association depends primarily on the so-called α-interaction domain (AID), 2The abbreviations used are: AID, α-interaction domain; cRNA, complementary RNA; GST, glutathione S-transferase; HA, hemagglutinin; GV, normalized conductance. located within the intracellular loop joining the first and second repeats of the α1-subunit. Secondary binding sites have been identified, but they appear to be specific to certain α1-β pairs, and they modulate particular aspects of channel function (7Dolphin A.C. J. Bioenerg. Biomembr. 2003; 35: 599-620Crossref PubMed Scopus (313) Google Scholar, 8Arikkath J. Campbell K.P. Curr. Opin. Neurobiol. 2003; 13: 298-307Crossref PubMed Scopus (423) Google Scholar). Recent work indicates that binding of a single β-subunit recapitulates function (9Dalton S. Takahashi S.X. Miriyala J. Colecraft H.M. J. Physiol. 2005; 567: 757-769Crossref PubMed Scopus (40) Google Scholar). Hence, it seems likely that β-subunit-binding sites available at the plasma membrane arise from unbinding of the β-subunit to the AID site during channel trafficking. This would imply a reversible interaction rather than a stoichiometric association, as proposed by Tareilus et al. (2Tareilus E. Roux M. Qin N. Olcese R. Zhou J.M. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1703-1708Crossref PubMed Scopus (154) Google Scholar). Here, we purified the β2a-subunit isoform, injected in oocytes expressing α1-subunit, and measured ionic and gating currents. Gating currents were correlated to the number of channels in the membrane by immunoassay. Using this approach we demonstrate that the α1-β-subunit interaction is dynamic and can occur at the plasma membrane. Binding to mature channels is reversible, and association of the β-subunit to the AID site is an absolute requirement to modulate channel function. Protein Preparation, Binding Assay, and Mutagenesis—The cDNA encoding the rat β2a subunit (3Neely A. Wei X. Olcese R. Birnbaumer L. Stefani E. Science. 1993; 262: 575-578Crossref PubMed Scopus (203) Google Scholar) was subcloned by conventional PCR methods into pRSET vector (Stratagene) to add an N-terminal polyhistidine tag (His6-β2a). The His6-β2a was expressed in BL-21 (DE-3) Escherichia coli bacteria by 2 h of induction with 0.5 mm isopropyl-β-d-thiogalactopyranoside at 37 °C and purified from the cleared cell lysate by metal affinity chromatography (Talon; BD Biosciences) followed by size exclusion chromatography on a Superdex™ S-200 column (Amersham Biosciences) pre-equilibrated with buffer containing 50 mm Tris buffer, 300 mm NaCl, 1 mm EDTA, pH 8.0. The fractions containing the protein were pooled, concentrated up to 2–4 mg/ml, and stored at –80 °C. Binding to AID was assayed as by Neely et al. (4Neely A. Garcia-Olivares J. Voswinkel S. Horstkott H. Hidalgo P. J. Biol. Chem. 2004; 279: 21689-21694Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), using a glutathione S-transferase (GST) fusion protein encoding the I-II loop of the CaV1.2 subunit (GST-AID), and as a negative control we used GST alone or fused to a 126-amino acid peptide derived from the C-terminal end of the chloride channel from human skeletal muscle ClC-1 (GST-ClC126). Oocyte Injections and Electrophysiological Recordings—Xenopus laevis oocytes were prepared, injected, and maintained as in previous report (4Neely A. Garcia-Olivares J. Voswinkel S. Horstkott H. Hidalgo P. J. Biol. Chem. 2004; 279: 21689-21694Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). All capped cRNA were synthesized using the MESSAGE-machine (Ambion, Austin, TX), resuspended in 10 μl of water and stored in 2-μl aliquots at –80 °C until use. The CaV1.2 subunit used in this study bears a deletion of 60 amino acids at the N-terminal end that increase expression (10Wei X. Neely A. Olcese R. Lang W. Stefani E. Birnbaumer L. Receptors Channels. 1996; 4: 205-215PubMed Google Scholar), and the CaV2.3 subunit corresponds to the human form (11Schneider T. Wei X. Olcese R. Costantin J.L. Neely A. Palade P. Perez-Reyes E. Qin N. Zhou J. Crawford G.D. Receptors Channels. 1994; 2: 255-270PubMed Google Scholar). The W470S mutation on the CaV1.2 subunit was incorporated by standard PCR methods. Electrophysiological recordings using the cut-open oocyte technique (12Taglialatela M. Toro L. Stefani E. Biophys. J. 1992; 61: 78-82Abstract Full Text PDF PubMed Scopus (127) Google Scholar) with a CA-1B amplifier (Dagan Corp., Minneapolis, MN) were performed 4–6 days after cRNA injection as described (4Neely A. Garcia-Olivares J. Voswinkel S. Horstkott H. Hidalgo P. J. Biol. Chem. 2004; 279: 21689-21694Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The external solution contained in mm, 10Ba 2+, 96 n-methylglucamine, and 10 HEPES, pH 7.0, and the internal solution 120 n-methylglucamine, 10 EGTA, and 10 HEPES, pH 7.0. For recording of oocytes expressing the CaV2.3 subunit, EGTA was replaced by BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′,-tetraacetic acid) in the internal solution for a better control of calcium-activated chloride currents. Data acquisition and analysis were performed using the pCLAMP system and software (Axon Instruments Inc., Foster City, CA). The currents were filtered at 2 kHz and digitized at 10 kHz. Linear components were eliminated by P/–4 prepulse protocol (3Neely A. Wei X. Olcese R. Birnbaumer L. Stefani E. Science. 1993; 262: 575-578Crossref PubMed Scopus (203) Google Scholar). For experiments with the purified β2a protein, the oocytes were injected with 50 nl of the protein stock solution 2–7 h before recordings. The final protein concentration was calculated assuming an oocyte volume of 525 nl that corresponds with a diameter of 1 mm. For bafilomycin treatment, the oocytes were exposed for 24 h to 500 nm concentration obtained from a 1 mm stock solution of bafilomycin A1 (Sigma-Aldrich) in Me2SO and compared with control oocytes incubated for the same period in 0.05% Me2SO. Surface Expression Measurements in Xenopus Oocytes—Surface expression of CaV1.2 was measured by immunoassay as described (13Altier C. Dubel S.J. Barrere C. Jarvis S.E. Stotz S.C. Spaetgens R.L. Scott J.D. Cornet V. De Waard M. Zamponi G.W. Nargeot J. Bourinet E. J. Biol. Chem. 2002; 277: 33598-33603Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The hemagglutinin (HA) epitope was inserted into the extracellular loop S5-H5 of domain II at residue 713 of the CaV1.2 subunit (Swiss-Prot P15381) by standard overlapping PCR using complementary oligonucleotides encoding the HA epitope and flanked with extra amino acids to yield CaV1.2-HA. 5–6 days after CaV1.2 RNA injection, the oocytes were separated into two groups: one for electrophysiological recordings and the other for immunoassay. Unless otherwise stated, all of the incubations for immunoassay were carried out at 4 °C. The oocytes were incubated in ND96 solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES, pH 7.6) with 1% bovine serum albumin (blocking buffer) for 30 min and then for 60 min in the presence of 1 μg/ml rat monoclonal anti-HA antibody (3F10; Roche Applied Science). After washes, the oocytes were incubated for 30 min with horseradish peroxidase-coupled secondary antibody (goat anti-rat FAB fragments; Jackson ImmunoResearch). Thereafter, the oocytes were extensively washed with blocking buffer and rinsed once in ND96 at room temperature. Individual oocytes were then placed in 50 μl of SuperSignal enzyme-linked immunosorbent assay femto substrate (Pierce) in 96-well microplates (Optiplate; PerkinElmer Life Sciences), and chemiluminescence was quantified 30 s later with a luminometer (Viktor2; PerkinElmer Life Sciences). Purified β2a Protein Binds to GST-AID Fusion Protein and Modulates CaV1.2 Channel Activity—The purified β2a elutes as a monodisperse peak from a size exclusion chromatography (Fig. 1A) and was capable of binding specifically to the CaV1.2-AID site because no binding activity was observed with GST alone or GST fused to an unrelated sequence (GST-ClC126; Fig. 1B). As in cRNA coexpression experiments (3Neely A. Wei X. Olcese R. Birnbaumer L. Stefani E. Science. 1993; 262: 575-578Crossref PubMed Scopus (203) Google Scholar, 14Perez Reyes E. Castellano A. Kim H.S. Bertrand P. Baggstrom E. Lacerda A.E. Wei X.Y. Birnbaumer L. J. Biol. Chem. 1992; 267: 1792-1797Abstract Full Text PDF PubMed Google Scholar), the purified β2a protein increases ionic currents and shifts the voltage dependence of calcium channel activation toward more negative potentials. Maximal barium current (IBa) was increased from –18 ± 4nA(CaV1.2 alone) to –184 ± 19 nA (CaV1.2/β2a protein), which compares with –101 ± 12 nA obtained from oocytes expressing CaV1.2/β2a cRNA (Fig. 1C). The reason for this difference is not clear. Channel expression depends on several factors, such as translation efficiency of cRNA and protein of the which of peak current our result the of the β2a The of channels during a to increasing was measured as the peak of the current during to peak currents were with to the to yield the (Fig. The of two was to each and the these are described in The difference when the effect of β2a as cRNA or as protein was that with the the relative of the first is ± than with CaV1.2/β2a cRNA ± This at and channels not by the auxiliary subunit when injected as a this we a by of a obtained with CaV1.2/β2a cRNA and of the one obtained β-subunit in Fig. This with the data obtained from oocytes injected with the CaV1.2/β2a protein ± S.E. of the of two that the normalized cRNA protein ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a in the of β-subunit a to gating currents that was followed by a current by the of (Fig. oocytes with CaV1.2/β2a cRNA ionic currents that were larger than gating currents (Fig. injection of the β2a protein to oocytes also to an increase in ionic current relative to gating currents (Fig. we the first 2 of the gating current by a from –80 to in Fig. and compared it with measured at the end of a to as described X. Neely A. Lacerda A.E. Olcese R. Birnbaumer L. Stefani E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Although in this of be because currents are from gating the is the same for β2a cRNA or β2a protein, because voltage dependence of activation is for both subunit Fig. of the for the three channel subunit a of are when CaV1.2 was expressed as in an ratio of ± the β2a-subunit is in this ratio is severalfold larger ± for CaV1.2/β2a protein and ± for CaV1.2/β2a All of the obtained from CaV1.2/β2a protein oocytes within the of the analysis from CaV1.2/β2a cRNA, that both of β2a appear in ionic currents at The of β2a Protein in with but the AID increase in ionic currents injection of β2a in a the gating currents remain more or This that CaV1.2 subunits in the plasma membrane are to the injected β2a this we oocytes with bafilomycin, an of that the traffic of intracellular S. K. J. Biol. 1997; PubMed Google Scholar). This is expected to the of CaV1.2 subunits into the plasma membrane and a of channel density by that membrane proteins at a rather that bafilomycin was the of synthesized surface expression was assayed by two independent immunoassay and charge movement during a voltage to (Fig. and were by more than by bafilomycin, and this was not injection of β2a For oocytes was ± which compares with ± measured in the of chemiluminescence were with ± or ± β2a in the bafilomycin our channel into plasma membrane. β2a with channels before they the plasma then injection of β2a protein not the function of calcium channels in oocytes. However, increases from ± to ± by injection of β2a protein in oocytes (Fig. and that β2a with channels in the plasma membrane. modulation of function of mature channels depends on an intact AID site and a mutation in CaV1.2 to a mutation that binding of the β-subunit in Waard M. Scott M. Campbell K.P. 1996; PubMed Scopus Google Scholar). this mutation modulation of function as well as binding L. H. G. L. Biophys. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). expression be by of the AID we the of CaV1.2 the W470S mutation of the plasma membrane by immunoassay and gating current (Fig. measured at with chemiluminescence a (Fig. also show that the within the AID sequence in CaV1.2 binding of β2a protein to a peptide this mutation (Fig. injected into oocytes, the β2a protein is no capable of increasing ionic currents (Fig. and of the voltage dependence (Fig. For oocytes, was ± which is to ± in oocytes demonstrating that modulation of function by the β-subunit an intact AID site. The W470S channels a ratio than an on the of the channel to in channel function with on within the AID domain in the of β-subunit have been L. A. A. L. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). β2a Protein of to the α1-β interaction is reversible, we the of β2a to inactivation in oocytes CaV2.3/β1b cRNA and it with effects on oocytes expressing CaV2.3 from oocytes expressing CaV2.3 cRNA by in a (Fig. when β2a cRNA is with CaV2.3 cRNA, inactivation several and a of of the peak current R. Qin N. T. Neely A. Wei X. Stefani E. Birnbaumer L. 1994; 13: Full Text PDF PubMed Scopus Google Scholar). of β2a protein on oocytes also inactivation (Fig. and increases from ± s for CaV2.3 alone to ± s This is than obtained from oocytes injected with both subunits as ± the β2a protein was also capable of increasing in oocytes the CaV2.3/β1b from ± s to ± The inactivation is also by β2a-subunit R. Qin N. T. Neely A. Wei X. Stefani E. Birnbaumer L. 1994; 13: Full Text PDF PubMed Scopus Google Scholar). is for CaV2.3 alone or with a with we injected β2a protein in oocytes expressing CaV2.3 alone or the CaV2.3/β1b voltage for was to the and a of in both (Fig. and also 2 for ± S.E. of the and the of current that inactivation for the subunit ± ± ± ± ± ± ± ± ± ± ± ± β2a ± ± ± in a The of inactivation by β2a protein in CaV2.3/β1b channel complexes arise by competitive or by modulation through a second binding site. both we the of inactivation by at of β2a (Fig. CaV2.3/β1b channel increase in concentration of β2a than CaV2.3 alone to increase β2a concentration channels of the presence of ± and ± for CaV2.3 alone or with These results suggest a competitive of by the of channel we the voltage dependence of inactivation of CaV2.3 and CaV2.3/β1b channels exposed to of β2a protein (Fig. subunit is expected to to a of a particular oocytes expressing a of subunit corresponds to the of the inactivation of and channels and Fig. The three were obtained by to from oocytes with the subunit this approach we the relative of the channel complexes and the of β2a protein these we obtained a of for β2a on oocytes and in CaV2.3/β1b in the maximal the relative of CaV2.3/β1b channels with increasing concentration of β2a protein when is (Fig. also that in oocytes the this of inactivation was not these results the of a competitive and The data show that the α1-β-subunit interaction to modulation of channel activity can take place at the plasma is reversible, and depends on an intact AID site. The was shown by a approach that of the effect of two β-subunit and on the inactivation of CaV2.3 channels. a of the voltage dependence of the inactivation of the subunit we the in intact CaV2.3/β1b channels were converted to channels with a of channels the addition of β2a protein in a that was with the of β-subunits by the presence of shifts the a in the maximal effect when compared with CaV2.3 our data are with a competitive of inactivation of CaV2.3/β1b channel complexes by β2a Although our analysis not a second binding for a single site from experiments that a to CaV1.2 recapitulates the effect of the auxiliary subunit on expression and function and no to coexpression of the β-subunit (9Dalton S. Takahashi S.X. Miriyala J. Colecraft H.M. J. Physiol. 2005; 567: 757-769Crossref PubMed Scopus (40) Google Scholar). our with both subunit yield one and two (Fig. increase the such as not in the 5 h that protein injection and electrophysiological recordings. to our the activity of the purified β-subunit protein (4Neely A. Garcia-Olivares J. Voswinkel S. Horstkott H. Hidalgo P. J. Biol. Chem. 2004; 279: 21689-21694Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The we measured in intact cells are larger than for in binding to GST-AID fusion for the β-subunit be for channels in the cell surface than in of protein and as proposed by and (2Tareilus E. Roux M. Qin N. Olcese R. Zhou J.M. Stefani E. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1703-1708Crossref PubMed Scopus (154) Google Scholar). in the is also with a reversible binding of the β-subunit that in to the of an stoichiometric This is to other intracellular regulatory the AID site. For protein binds to a site the AID the β-subunit, and and calcium channel A.C. Rev. 2003; PubMed Scopus Google Scholar, G. X. N. M. A. De Waard M. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). issue that remains rather is our that a with β-subunit binding and well in the plasma as binding of the β-subunit to the AID site were not for surface be that the of the binding to the AID to the that is no in and on but it is to the β-subunit to the in the of experiments J.M. Kim J. Biol. 2005; PubMed Scopus Google Scholar) that can also the plasma membrane the AID appears not to be as as and the β-subunit traffic through the by binding to other sites of the α1-subunit. the CaV1.2 N-terminal used in this study the from the AID of channel expression on the have results for other β-subunit (4Neely A. Garcia-Olivares J. Voswinkel S. Horstkott H. Hidalgo P. J. Biol. Chem. 2004; 279: 21689-21694Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). This would also N-terminal deletion (10Wei X. Neely A. Olcese R. Lang W. Stefani E. Birnbaumer L. Receptors Channels. 1996; 4: 205-215PubMed Google Scholar). we demonstrate that the interaction the β-subunit and the pore-forming subunit of voltage-gated calcium channels is The dynamic nature of this interaction for modulation of channel function. this interaction the AID proposed to control of the channel to the plasma membrane. and for