©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Roles of a Membrane-localized Subunit in the Formation and Targeting of Functional L-type Ca Channels (*)

(Received for publication, August 23, 1995; and in revised form, October 6, 1995)

Andy J. Chien (1)(§) Xiaolan Zhao (1) Roman E. Shirokov (2)(¶) Tipu S. Puri (1) Chan Fong Chang (1) Dandan Sun (1) Eduardo Rios (2) M. Marlene Hosey (1)(**)

From the  (1)Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611 and the (2)Department of Physiology, Rush University, Chicago, Illinois 60612

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We report several unexpected findings that provide novel insights into the properties and interactions of the alpha(1) and beta subunits of dihydropyridine-sensitive L-type channels. First, the beta subunit was expressed as multiple species of 68-72 kDa; the 70-72-kDa species arose from post-translational modification. Second, cell fractionation and immunocytochemical studies indicated that the hydrophilic beta subunit, when expressed alone, was membrane-localized. Third, the beta subunit increased the membrane localization of the alpha(1) subunit and the number of cells expressing L-type Ca currents, without affecting the total amount of the expressed alpha subunit. Expression of maximal currents in alpha/beta co-transfected cells paralleled the time course of expression of the beta subunit. Taken together, these results suggest that the beta subunit plays multiple roles in the formation, stabilization, targeting, and modulation of L-type channels.


INTRODUCTION

Calcium channels are multisubunit proteins that minimally consist of alpha(1), alpha(2), and beta subunits(1, 2) . The alpha(1) subunit is the pore-forming channel subunit and is the ``signature'' subunit that determines many of the basic characteristics of the different types of Ca channels. The roles of the ``accessory'' subunits are less certain, and much remains to be revealed about the roles of the accessory subunits in Ca channel formation, processing, regulation, and function. Co-expression of the alpha(2) and/or beta subunits clearly results in larger Ca currents from expressed alpha(1) subunits(3, 4, 5, 6, 7, 8, 9) , and several reports have demonstrated that the beta subunits can modulate the activation and inactivation kinetics of the channels(3, 4, 5, 7, 8, 10, 11) . While channel-specific ``beta'' subunits appear to play roles in certain Na and K channels, none of these channels are as complex as the Ca channels. The beta subunits are particularly intriguing since they are very hydrophilic and are not predicted to possess membrane-spanning domains. To begin to address how these proteins participate in channel function, we have analyzed the properties and interactions of the alpha subunit and beta subunits of Ca channels in transiently transfected human embryonic kidney (HEK) (^1)cells. The particular alpha and beta subunits chosen for analysis are expressed in the heart, brain and other tissues. Based on mRNA studies, they appear to be critical components of what has been traditionally referred to as the ``cardiac'' L-type channel, and variants of these subunits are likely to also comprise ``brain'' L-type channels. Our results reveal previously unknown characteristics of the proteins and their interactions, and suggest that the beta subunit plays multiple roles in the formation, stabilization, targeting, and modulation of the channel complex. In addition, these are the first results to characterize the properties of these rare membrane proteins at the biochemical level and provide new insights into the poorly understood properties of these proteins.


EXPERIMENTAL PROCEDURES

Materials

The alpha cDNA (12) and the beta cDNA (5) were generous gifts from Drs. Ed Perez-Reyes, Chris Wei, and Lutz Birnbaumer (formerly at Baylor College of Medicine). The human embryonic kidney (HEK) 293 cells and the pBRG4 vector were gifts of Dr. Ron Kopito (Stanford University). The large T-antigen-transformed HEK cells (tsA201 cells) were a gift from Dr. Richard Horn (Thomas Jefferson University). The peptide used to prepare the CardC antisera was synthesized by Multiple Peptide Systems (San Diego, CA). FITC- and TRITC-coupled secondary antibodies for immunocytofluorescence were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Protein A-agarose and protein G-agarose were purchased from Sigma. All other reagents were from standard sources. [S]methionine and [^3H]PN200-110 were purchased from Amersham Corp.

Preparation of Expression Vectors and Transfection Procedures

The cDNA sequence for alpha was excised from pGEM3-alpha using HindIII and KpnI, subsequently blunt-ended and cloned into a blunt-ended EcoRI site of the pRBG4 polylinker. The cDNA sequence of beta was excised from pKS-beta using EcoRI and cloned into the EcoRI site of the pRBG4 polylinker and the EcoRI site of pMT21. The cDNA for the epitope-tagged beta, which contains the epitope TPPPEPET immediately distal to the last amino acid residue in beta (Q604), was made using the following primers (KT(3) coding sequence in plus strand shown underlined): 5`-GTATACATCCGCCAAACTCCACCACCAGAACCAGAAACATGACCGTGCGTGTCTCTGC-3` (plus strand) and 5`-TTGGACAAACCACAACTAGAATGC-3` (minus strand). The fragment encoding the KT(3) epitope was substituted into pRBG-beta as an AccI-HindIII fragment.

Cell lines were maintained in DMEM (Life Technologies, Inc.) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO(2). Transfections were done using the HEPES-buffered calcium phosphate method (13) in 100-mm polystyrene tissue culture plates. Cells were refed with fresh media 1-3 h prior to addition of DNA. Unless otherwise indicated in the text, transfections were performed with 30 µg of each expression plasmid, with all transfections normalized to 60 µg of total DNA using either pRBG4 vector or sheared salmon sperm DNA (Life Technologies, Inc.). Cells were incubated with DNA for 6-8 h, after which a 30% DMEM-Me(2)SO solution was added to a final concentration of 10% Me(2)SO, followed by a 6-min incubation. Subsequently, cells were refed with fresh media and incubated at 37 °C until further evaluation. Post-transfection times reflect hours following Me(2)SO shock, which is considered as 0 h. In co-transfection experiments, similar results were seen when DNA amounts were normalized with either salmon sperm DNA or pRBG4 vector or when DNA amounts were not normalized (data not shown). Experiments using cycloheximide contained cycloheximide at a concentration of 100 µg/ml.

Antibody Preparation and Immunoblotting

The Card C antibody was prepared in rabbits against a peptide C (QSEEALADRRAGVS; residues 2156-2169) in the C-terminal portion of the predicted protein sequence (Fig. 1A). The antipeptide antibody was raised and characterized in our laboratory (^2)using methods previously described(14) . The internal alpha antibody was generated against a fusion protein containing sequence from the II-III cytoplasmic linker (residues 812-929) of the predicted alpha sequence (Fig. 1A), which was amplified using PCR and cloned in-frame into an expression vector encoding a 6x-His tag on the N terminus (Qiagen Corp., Chatsworth, CA). The PCR primers used contained the sequence (forward primer) 5`-CCGGAGAAGAAACAAGAGGTGG-3` and (reverse primer) 5`-ACGATACGGTGACACTGGAGGC-3`. The antibody against the beta subunit was prepared against a fusion protein containing the C-terminal portion (residues 462-600) of the predicted beta sequence (Fig. 1C). The C terminus of beta was amplified by PCR and cloned in-frame into an expression vector encoding a 6x-His tag on the N terminus. The PCR primers used contained the sequences 5`-GAAGAAGAACCTTGTCTGGAACC-3` (forward primer) and 5`-TACATCCCTGTTCCACTCGCCAGC-3` (reverse primer). Fusion proteins were purified using a Ni-NTA agarose resin (Qiagen Corp). Following purification of the fusion proteins, they were injected into rabbits and polyclonal antisera were prepared (Bethyl Laboratories, Montgomery, TX). Antibody production was monitored by either an enzyme-linked immunosorbent assay using the alpha(1) peptide as the antigen, or by using fusion proteins and baculovirus-expressed proteins^2 in SDS-polyacrylamide gel electrophoresis/immunoblotting assays. The specificity of the beta antibodies was assessed using the beta fusion protein and membrane preparations containing different types of beta-subunits (Fig. 1). In contrast to its reactivity toward the beta subunit, the antibody showed no reactivity toward transverse-tubule membranes prepared from rabbit skeletal muscle (14) which contain the beta(1) subunit (Fig. 1).


Figure 1: Characterization of expressed alpha and beta calcium channel subunits in transiently transfected HEK cells using subunit-specific antisera. A, a linear representation of the alpha protein delineating the epitopes used to generate the Card C and Card I antisera, as described under ``Experimental Procedures.'' B, detection of the expressed alpha protein using immune and preimmune antisera for Card C and Card I. The figure shows immunoreactivity against membranes from non-transfected HEK cells (lanes 1, 3, 6, and 8) and membranes from transiently transfected alpha cells (lanes 2, 4, 5, and 7). Immune Card C and Card I antisera both recognized a protein in transfected cells with a relative mobility of 240 kDa. C, linear representation of the beta(2) protein, delineating the location of the two regions conserved among known beta isoforms, as well as the region in the C terminus used to generate the beta(2) antisera as described under ``Experimental Procedures.'' D, detection of the expressed beta subunit by the beta(2) antisera in membranes isolated from transiently transfected beta cells (lane 1), non-transfected cells (lane 2), and transverse tubule membranes purified from skeletal muscle (lane 3). Samples were electrophoresed on a 8% polyacrylamide gel.



To visualize expressed proteins in preparations from transfected cells by immunoblotting, either whole cells or crude membranes were used. Cells were homogenized in 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA and protease inhibitors (0.2 mM phenylmethysulfonyl fluoride, 100 nM aprotinin, 1 µM leupeptin, 1 µM pepstatin A, 1 mM soybean trypsin inhibitor, and 1 mM iodoacetamide) using a Tri-R homogenizer at setting 7. To prepare crude membranes, the homogenates were centrifuged at 2100 rpm for 5 min and the supernatants were subsequently centrifuged at 42,000 rpm for 60 min in a Type 65 rotor. Proteins were separated on SDS-polyacrylamide gels containing the indicated concentrations of acrylamide, transferred to nitrocellulose, and processed for immunoblotting as described previously(14) . Detection was with enhanced chemiluminescence (Amersham), horseradish peroxidase, or alkaline phosphatase as described(15) .

Immunofluorescence Studies

Cells for immunofluorescent studies were transfected in 100-mm plates as described above and subsequently split 20 h post-transfection to coverslips coated with poly-L-lysine (Sigma). Cells were fixed with 4% paraformaldehyde (EM Sciences) 40-60 h post-transfection and subsequently permeabilized with 0.5% Triton X-100 at room temperature. Cells were then incubated with normal goat serum (Sigma) for 1 h at room temperature, followed by incubation with the primary antibody for 1 h at room temperature, and then incubation with an TRITC- or FITC-coupled secondary antibody for 1 h at room temperature. Coverslips were mounted onto slides for viewing with confocal microscope.

Intact-cell Radioligand Binding

Cells were removed from tissue culture dishes using DMEM/F-12 (JRH Biosciences, Lenexa, KS) and gentle pipetting. Cells were resuspended at a concentration of 0.2-0.5 mg of protein/ml. The radioligand used was [^3H]PN200-110. Nonspecific binding was assessed in the presence of 10M nitrendipine. Whole-cell protein assays were used to determine the amount of protein per assay (20-50 µg). More than 80% of the cells used for binding assays excluded trypan blue. Binding reactions were incubated for 3 h at 4 °C and subsequently filtered through Whatman GF/A filters using a Brandel cell harvester. Scatchard analysis was performed to assess B(max) and K(d). Statistical calculations were performed using the GraphPad InStat program.

Pulse-Chase Determinations

Transfected cells were incubated with methionine-free media (Life Technologies, Inc.) for 30 min prior to labeling, and subsequently incubated with 2-3 ml of methionine-free DMEM containing [S]methionine (0.1 mCi/ml) for 30 min at 37 °C and chased using media containing an excess of unlabeled methionine (15 mg/ml). Cells were harvested at various time points following the removal of the radiolabeled media and subsequently frozen at -80 °C. Cells were thawed at 4 °C, and membranes were prepared as described above. Membrane particulate fractions were solubilized using 1% digitonin and 1% Nonidet P-40 in homogenization buffer containing protease inhibitors. Solubilized proteins were immunoprecipitated using either the Card C antibody coupled to protein A agarose, the Card I antibody coupled to protein G-agarose, or the beta(2) antibody coupled to protein A-agarose. Immunoprecipitated proteins were eluted with SDS-polyacrylamide gel electrophoresis loading buffer for 2-5 min at 95 °C. Radiolabeled proteins were visualized using FujiBAS imaging plates and a BAS2000 phosphorimager.

Whole-cell Patch-clamp of Transfected HEK Cells

Cells were split 8 h post-transfection onto poly-L-lysine-coated coverslips. Voltage clamping, pulse generation, and data acquisition were carried out with an Axopatch 200A or 1A series voltage clamp (Axon Instruments, Inc., Foster City, CA) and a 16-bit A/D-D/A card operating at 120 kHz (HSDAS 16, Analogic Corp., Wakefield, MA), with a PC-compatible computer. The data acquisition and pulse generation routines were generated by Dr. Ivan Stavrovsky in the Rios laboratory. Asymmetric currents were obtained by subtraction of control currents elicited with pulses from -130 to -100 mV. The set of control and test pulses were separated by 20 s. The internal solution contained (in mM) 150 Cs, 125 Asp, 15 Cl, 5 MgATP, 10 HEPES, 10 EGTA, pH 7.6, adjusted by CsOH. The external solution contained (in mM) 130 NaCl, 10 HEPES, and either 10 Ca or 10 Ba. When necessary, we added 10M of TTX in the external solution in order to block endogenous Na inward current. The density of this current was relatively small (less than 1 A/F) and early passage cells did not have this Na current. Differences in current densities in different groups of cells were analyzed using the Mann-Whitney rank sum test and forward stepwise regression routines of the SigmaStat software package (Jandel Scientific, Corte Madera, CA).


RESULTS

Transiently Transfected Cells Express a Full-length alpha Protein

We first asked if the alpha subunit could be expressed in HEK cells using the pRBG4 mammalian cell expression vector which had previously been used to express the µI Na channel in HEK cells(16) . Cells were transiently transfected with either empty vector or the pRBG-alpha plasmid using the calcium phosphate method and isolated after 40-60 h as described under ``Experimental Procedures.''

Two different antibodies specific for the alpha subunit, Card I and Card C (Fig. 1A), were developed and used to visualize the expressed protein on immunoblots. The alpha subunit was detected as a single large molecular species of 240 kDa, which migrated more slowly than the myosin (205 kDa) molecular mass marker (Fig. 1B). The observed size of the alpha subunit is close to the cDNA-predicted size of the full-length alpha subunit (242,771 daltons; (12) ). The size of the expressed protein and its reactivity with the Card C antibody, which is directed at 14 of the last 16 amino acids in the C terminus, suggested that the expressed protein was a full-length protein with an intact C terminus. In addition, a single immunoreactive protein of the same electrophoretic mobility was detected by the Card I antibody (Fig. 1B), which reacts with the II-III cytoplasmic linker of the cardiac alpha(1) subunit, thus providing further evidence that the expressed alpha(1) subunit is an intact full-length protein. In agreement with this conclusion, we also observed expression of a similarly sized protein using recombinant baculovirus to direct the expression of the alpha subunit in insect Sf9 cells.^2 The baculovirus-expressed protein additionally contained an epitope tag in its N terminus, which allowed us to confirm that the expressed protein was full-length. The size of the expressed alpha subunit was larger than that of the alpha(1) subunit in L-type channel preparations previously purified from cardiac tissue(17) . This suggests that the purified cardiac protein may have been proteolyzed during the isolation procedure, or alternatively, that tissue-specific modification of the alpha(1) subunits occurs. Previous observations have shown that C-terminal truncation of other alpha(1) subunits occurs in native tissues such as skeletal muscle (14, 18) and brain(19, 20) .

The beta Subunit Is Expressed as Multiple Forms and Is Post-translationally Modified

The expressed beta subunit was visualized on immunoblots using an antibody specific for the C-terminal region of beta (Fig. 1, C and D). The anti-beta antibody detected a protein of 68 and 72 kDa in transfected HEK cells (Fig. 1D, lane 1). The cDNA for beta predicts a highly hydrophilic protein of 68,191 daltons, which is approximately 10 kDa larger than the beta(1) subunit expressed in skeletal muscle(5, 6, 21, 22) . The specificity of the beta antibody was demonstrated by the lack of detection of immunoreactive species in non-transfected cells or in purified transverse tubule membranes from skeletal muscle, which contain the beta(1) subunit (Fig. 1D, lanes 2 and 3). When higher resolution SDS gels were used, the expressed beta subunit was detected in immunoblots as a series of three to four immunoreactive bands between 68 and 72 kDa (Fig. 2A). These multiple immunoreactive species were also detected when cells were labeled with [S]methionine and the beta subunit was immunoprecipitated with the anti-beta antibody (Fig. 2A, lane 1). The fastest migrating species corresponded to the predicted 68-kDa size, while the slower migrating species were larger than predicted, possibly representing post-translationally modified proteins. Expression of beta, which contains the entire coding sequence of beta in addition to an 8-amino acid C-terminal KT(3) epitope tag(23) , also led to the appearance of multiple immunoreactive species that could be detected with either the anti-beta antibody or with the monoclonal KT(3) antibody (Fig. 2B). The comparative electrophoretic mobility of beta, which has a predicted molecular mass of 69,183 daltons, suggested that the difference in size between the two fastest migrating species of untagged beta was less than 1 kDa (Fig. 2A, lane 3). The reactivity of all the beta isoforms with a monoclonal KT(3) antibody (Fig. 2B) indicates that the multiple isoforms do not represent C-terminal truncations. Consequently, it is unlikely that modifications involving proteolytic processing of the C terminus, such as prenylations, are involved in the post-translational modification of the beta subunit. To our knowledge, this is the first demonstration of the protein product of the beta cDNA.


Figure 2: Multiple species and pulse-chase analysis of beta subunit synthesis in transiently transfected HEK cells. A, higher resolution of the beta protein on a 6% polyacrylamide, 24-cm gel reveals multiple immunoreactive species on a Western blot, ranging from 68 to 72 kDa in size (lane 2). A phosphorimage of the beta subunit immunoprecipitated from cells metabolically labeled with [S]methionine revealed at least three labeled bands ranging from 68 to 72 kDa (lane 1). Comparison of immunoreactive staining of membranes from beta cells with membranes from cells expressing the epitope-tagged beta subunit (lane 3), which contains an additional 8 amino acid residues, suggests that the relative molecular masses of the lower doublet are 68 and 69 kDa. The arrow marked 80 kDa represents the position of the prestained albumin molecular mass marker. B, the beta antisera and the monoclonal KT(3) antibody had the same immunoreactive profile against membrane particulate fractions from large T-transformed HEK (tsA201) cells expressing the epitope-tagged beta subunit. The KT(3) antibody also reacted with the 82-kDa large T antigen, which contains the KT(3) epitope. C, labeling of transiently transfected beta cells with [S]methionine, followed by solubilization and immunoprecipitation with the anti-beta antibody, revealed at least three distinct isoforms of the beta subunit. Shown are S-phosphorimages of the beta subunit immunoprecipitated from metabolically labeled beta (top panel) and alpha/beta (bottom panel) cells. The beta subunit was initially synthesized as a 68-kDa nascent protein, which was subsequently modified to at least two higher molecular mass isoforms indicated by the filled arrows.



Pulse-chase experiments using [S]methionine were performed using transfected beta cells to determine whether the higher molecular mass isoforms were the result of post-translational modification. The results indicated that the beta protein was initially synthesized as a 68-kDa protein (Fig. 2C), the size predicted from the cDNA sequence. As time elapsed, higher molecular mass bands of 69 and 72 kDa appeared on the S image, suggesting that the higher molecular mass isoforms of the beta subunit arose due to multiple post-translational modifications of the 68-kDa form. Similar results were observed in cells expressing either beta alone (Fig. 2C, upper panel) or both alpha(1) and beta (Fig. 2C, lower panel). The modification was not due to phosphorylation of the protein (^3)and the predicted primary structure lacks signature sequences, in the proper context, for myristoylation or isoprenylation. The modification took place on a relatively slow time scale; the change in distribution of isoforms was not observed until several hours after the initial synthesis of the protein. Co-expression with alpha did not appear to affect either the rate or extent of the post-translational modification. Further studies will be necessary to reveal the nature of these modifications.

The beta subunit isoforms, measured collectively, appeared to turn over relatively slowly with no discernible difference between beta and alpha/beta cells. When the total immunoreactive beta subunit bands were analyzed collectively, the estimated half-life was >8 h (data not shown). However, this is a crude estimate, because the change in distribution of label between the multiple forms, as well as the possibility that the different isoforms may turnover at different rates, did not allow an accurate analysis of the half-life.

The Hydrophilic beta Subunit Is Localized to Membranes

Because the beta subunits of Ca channels are very hydrophilic and possess no predicted transmembrane domains, they might be expected to behave as soluble cytosolic proteins, particularly when expressed in the absence of other channel subunits. Against this expectation, when the amount of immunoreactive beta protein was assessed in cytosolic and crude membrane fractions, all immunoreactive material was found in the particulate fractions (Fig. 3A). No immunoreactivity was detected in cytosolic fractions (Fig. 3A). Further characterization of the properties of the membrane localized beta subunit were performed to assess the solubility of the beta subunit in salt and in various detergents (Fig. 3B). All agents tested solubilized a portion of the protein. The highest degree of solubility was observed in 1% digitonin/0.4 M NaCl, which was comparable to that observed with 1% SDS (data not shown). Significantly less protein was solubilized with Nonidet P-40 or Triton X-100, which may suggest an association of one or more forms of beta with cytoskeletal elements. NaCl also appeared to solubilize a portion of the protein. Interestingly, beta protein from fractions that were salt-insoluble could be further solubilized with digitonin, and beta protein from digitonin-insoluble fractions could be further solubilized with NaCl (data not shown), suggesting that salt and digitonin may solubilize two distinct populations of beta. This explanation could account for the high solubility of beta in digitonin/NaCl despite relatively poorer solubility in digitonin alone and NaCl alone.


Figure 3: Cellular distribution of the beta subunit expressed in transiently transfected HEK cells. A, levels of the beta protein were evaluated in membrane particulate fractions (lanes 1 and 2) and cytosolic fractions (lanes 3 and 4). The beta(2) protein was localized to membrane particulate fractions both in transiently transfected beta cells (lanes 1 and 3) and alpha/beta cells (lanes 2 and 4). B, solubilization of the beta protein in detergents and in salt as described under ``Experimental Procedures.'' Shown are immunoblots of supernatants (top panel) and pellets (bottom panel) following solubilization of 100 µg of membrane particulate fraction in various detergents and NaCl.



To further characterize the cellular location of the expressed beta subunit, cells transiently expressing the beta subunit were fixed, permeabilized, stained with the beta(2) antisera, and viewed using confocal microscopy (Fig. 4). In these experiments, staining of the beta subunit clearly was present at the periphery of the cells, suggesting a membrane localization (Fig. 4A). In addition, diffuse staining was observed in intracellular compartments. No specific immunoreactivity was seen when preimmune antisera was used (data not shown). Based on the cellular fractionation data, the intracellular pools of the beta subunit must reflect beta in intracellular membranes associated with protein processing rather than ``soluble'' protein. Indeed, when cells were pretreated with cycloheximide 2 h prior to fixing and staining, almost all of the beta-subunit immunofluorescence was seen in dense patches at the plasma membrane, with very little staining inside the cell (Fig. 4C). Presumably, cycloheximide treatment inhibited new synthesis of the beta subunit and allowed the protein that was already made to be processed and transported to the plasma membrane. These results strongly suggest that the beta subunit, which has been referred to as ``the cytosolic subunit,'' is actually localized to membranes. The beta subunit was also membrane-localized when expressed in mammalian baby hamster kidney cell (^4)and in insect Sf9 cells from a recombinant baculovirus,^2 indicating that this is not a cell-specific phenomenon.


Figure 4: Whole-cell immunofluorescence of transiently transfected beta cells. Cells were transiently transfected with beta subunit and fixed with paraformaldehyde at 40 h post-transfection. Shown are confocal microscopy (A and C) and phase-contrast (B and D) views of cells stained with the beta(2) antibody. For panels C and D, cells were pretreated with cycloheximide, as described in the text. Strong immunoreactivity to the beta(2) antibody was detected at the plasma membrane with diffuse cytosolic staining. Treatment with cycloheximide eliminates the diffuse cytosolic staining, further enhancing the visualization of beta(2) staining at the plasma membrane.



Expression of the beta Subunit Increases the Membrane Localization of the alpha(1) Subunit

Previous studies have shown that the beta subunit increases current density, but the mechanism underlying this phenomenon is not understood. Therefore, we next asked whether the beta subunit might modulate the levels of the alpha(1) subunit and/or its membrane localization. Upon cell fractionation, the alpha subunit was localized to particulate fractions (Fig. 5A, inset). Importantly, co-expression with the beta subunit did not affect the total amount of detectable alpha(1) subunit. This was ascertained by immunoblotting of membranes and/or cell lysates (Fig. 5A) in a large number of preparations. In addition, we observed that peak alpha(1) expression occurred between 24 and 36 h post-transfection in both alpha (data not shown) and alpha/beta cells (Fig. 8C). No significant differences in total alpha protein levels were seen at any time point between alpha and alpha/beta cells within individual experiments.


Figure 5: Cellular distribution of the alpha subunit in transiently transfected HEK cells. A, relative levels of expression of the alpha protein in transiently transfected alpha and alpha/beta cells. Inset shows alpha immunoreactivity in whole-cell lysates (inset, lane 1) and membrane particulate fractions (inset, lane 2), and no immunoreactivity in cytosolic fractions (inset, lane 3). The results are the average of five independent experiments. B and C, whole-cell immunofluorescence of transiently transfected alpha (B) and alpha/beta cells (C). Shown are phase-contrast and confocal microscopy views of cells stained with the Card C antibody. In B, note that the staining is predominantly intracellular and perinuclear. Upon co-transfection with beta, staining of alpha becomes localized to the plasma membrane (C). Arrows are included for reference.




Figure 8: Effects of the beta subunit on the appearance of Ca currents as a function of time post-transfection. A, representative traces from transiently transfected alpha and alpha/beta cells at different times post-transfection. Trace a (15 h) is from an alpha cell, while traces b (15 h), c (42 h), d (70 h), and e (95 h) are from alpha/beta cells. Pulse protocol is shown on top with voltages indicated in millivolts. B, peak current density in alpha (closed bars) and alpha/beta (open bars) cells at specific times post-transfection, obtained using whole-cell patch-clamp. Each bar represents an average of 3-4 alpha cells or 5-8 alpha/beta cells, measured during 2-h intervals beginning at the following times post-transfection: 15, 23, 33, 38, 44, 50, 70, and 95 h. C, representative immunoblots of membranes from transiently transfected cells, analyzed for subunit expression at specific times post-transfection using subunit-specific antibodies. No significant difference in alpha levels was detected between alpha and alpha/beta cells (Fig. 5). Note that the staining patterns of the blot cannot be used to measure the absolute amount of either expressed protein (i.e. the data cannot be analyzed to determine the ratios of subunit protein levels).



While the beta subunit did not increase the total alpha protein detected (Fig. 5A), the beta subunit did affect the cellular localization of the alpha(1) subunit. Cells transfected with alpha alone and stained with the Card C antibody yielded immunostaining that was largely perinuclear, and very little staining of the plasma membrane was evident (Fig. 5B). Only a few percent (no more than 5%) of the cells exhibited weak, diffuse cytosolic or membrane staining in cells transfected with alpha alone (data not shown). In marked contrast, when cells were transiently transfected with both the alpha and beta subunits, there was significant membrane staining of alpha (Fig. 5C) suggesting that co-expression of alpha and beta subunits led to increased localization of the alpha(1) subunit to the plasma membrane. These results provide evidence that interactions between the alpha(1) and beta subunits may be critical in channel formation and/or channel targeting to the membrane.

In order to determine if the alpha and beta subunits were co-localized in alpha/beta cells, cells were co-transfected with alpha and beta, co-stained with the Card C and KT(3) antibodies, and visualized using FITC- and TRITC-conjugated secondary antibodies, respectively (Fig. 6). The patterns of fluorescein staining of the alpha subunit (Fig. 6, left panels) and rhodamine staining of the beta subunit (Fig. 6, center panels) are strikingly similar. In both cells, there is strong, punctate staining of the cell periphery, as well as some intracellular staining. When the fluorescein and rhodamine images were overlaid (Fig. 6, right panels), most of the membrane staining became yellow, indicating that the majority of the alpha(1) and beta subunits in the membrane were co-localized. Clear areas of intracellular staining of each subunit alone, particularly the alpha subunit, were also visible. In addition, small areas of membrane staining of alpha only or beta only were apparent, suggesting that a small percentage of each subunit might be present separately in the plasma membrane.


Figure 6: Co-localization of alpha and beta protein in transiently transfected alpha/beta cells. Cells were co-transfected with alpha and the epitope-tagged beta and subsequently co-stained with Card C and anti-KT(3) antibodies. Visualization was done using FITC- and TRITC-conjugated secondary antibodies. Fluorescein staining on the left panels shows the pattern of alpha expression, while rhodamine staining in the center panels indicates the pattern of beta expression. On the right panel, an overlay shows the co-localization of the staining for both alpha and beta.



Conceivably the beta subunit might stabilize the alpha(1) subunit and increase the probability that it localizes to the plasma membrane. Pulse-chase studies using [S]methionine were used to determine the half-life of the expressed alpha(1) subunit in transiently transfected alpha and alphabeta cells. Cells were initially labeled with [S]methionine 20-24 h post-transfection, fractions were collected every 2 h following the addition of the chase media, and were subsequently immunoprecipitated with subunit-specific antibodies. In both alpha and alpha/beta cells, the observed half-life of the alpha subunit was about 3 h (Fig. 7, A and B). The total pool of alpha protein also was visualized using immunoblotting (data not shown), which demonstrated that the decline in S-labeled protein was not due to declines in total levels of the protein. From these results, it appeared that the beta subunit did not change the rate of turnover of the alpha protein. However, because this analysis cannot differentiate between alpha(1) subunit present intracellularly and in the plasma membrane, we cannot rule out the possibility that the beta subunit may specifically stabilize the membrane-localized protein.


Figure 7: Pulse-chase analysis of alpha protein turnover in transiently transfected HEK cells. A, cells were labeled with S and analyzed in post chase studies as described under ``Experimental Procedures.'' The graph depicts the turnover of the alpha protein with respect to time in alpha (closed circles) and alpha/beta (open circles) cells. Data were obtained by quantifying the S signal using a phosphorimager. B, natural log of radioactive decay of the alpha protein in alpha and alpha/beta cells. Data from A were fitted to a single exponential term by linear least-squares regression analysis, corresponding to a predicted t of 3.45 h in cells expressing alpha alone (r = 0.96) and 3.12 h in cells expressing both alpha and beta (r = 0.98). Data for alpha cells represent averages of two experiments ± S.D., while data for alpha/beta cells represents the average of three experiments ± S.E.



Expression of the beta Subunit Is Necessary for Saturable Ligand Binding

Previous studies have demonstrated that the alpha(1) subunit contains the dihydropyridine (DHP) binding site(24, 25, 26, 27) . In order to assess whether further interactions between subunits could be identified in this expression system we characterized the ability of the expressed proteins to bind the DHP [^3H]PN200-110. Cells were transfected with either the alpha subunit alone or with both subunits and used in intact cell binding assays at 4 °C. Cells expressing alpha alone exhibited very low levels of binding which could not be accurately assessed for either B(max) or K(d) (n = 3). Expression of alpha in these cells was readily detected by immunoblotting using either Card I or Card C, indicating that the lack of binding was not due to lack of protein expression, but perhaps due to lack of proper protein targeting and/or protein conformation. In contrast, cells co-transfected with both alpha and beta exhibited readily detectable saturable binding with a B(max) of 0.22 ± 0.05 pmol/mg protein and a K(d) of 0.41 ± 0.10 nM (n = 6). The ability of transiently co-transfected cells to bind DHPs implies that co-expressed subunits in these cells form functional complexes at the plasma membrane, confirming that membrane localization of the channel in the HEK cells largely depends on the beta subunit.

The beta Subunit Increases Calcium Currents in a Timedependent Manner That Parallels the Time Course of Expression of the beta Subunit

Roles of the beta subunit on the membrane localization and functioning of L-type channels were also studied by measuring whole-cell Ca currents in transiently transfected cells, as described under ``Experimental Procedures.'' Several reports have demonstrated that the beta subunits increase peak current density(3, 4, 5, 6, 7, 8, 9) . We assessed the time course for this effect to occur in the transient transfection system used here. In order to assess these effects accurately, it was important to eliminate as much as possible variability in currents from different batches of transfected cells. Therefore, we split batches of cells and transfected them in parallel with either alpha or alpha/beta and then followed the time course of current expression by carrying out brief recording sessions in which we alternated measurements from alpha and alpha/beta cells at different times post-transfection. Peak currents, recorded at steps to 30 mV, are shown from a representative alpha cell (Fig. 8A, trace a) and alpha/beta cells (Fig. 8A, traces b-e) at different times after transfection. Currents were first detected 12-24 h after transfection in the presence or absence of the beta subunit. Co-expression with the beta subunit increased current density in a time-dependent manner (Fig. 8A). The average peak current densities as a function of time after transfection are shown in Fig. 8B. In alpha cells, Ca currents were small and similar in size for the first 40 h, after which they began to decline (Fig. 8B). Currents were about 2-4-fold larger in alpha/beta cells, reached a maximum at 40-50 h after transfection, and then decayed slowly, remaining sizable even after 5 days. As analyzed by the MannWhitney rank sum test, currents in alpha/beta cells were significantly larger (p < 0.05) than in alpha cells at any time from 14 to 95 h post-transfection. In alpha/beta cells, currents were significantly different between 14 and 44 h (p = 0.024) and between 44 and 95 h (p = 0.029). Although currents were not significantly different for intermediate points between 14 and 44 h, current density strongly correlated with time (R = 0.92) and could be predicted as a linear function of time (p = 0.027). In order to understand the basis for these changes, we compared the data to the time course of expression of the two subunits that we obtained by immunoblotting (Fig. 8C). The expression of the alpha-subunit detected with specific antibodies was high at 12 h and peaked at 24-36 h post-transfection, after which it declined and was significantly diminished by 60 h (Fig. 8C). In contrast, the expression of beta was low at 12-24 h but continued to increase up to 45 h, and remained relatively constant until 70 h (Fig. 8C). Taken together, the prolonged rising phase in the time course of peak current expression introduced by co-transfection with the beta subunit appears consistent with the slow increase in expression of the beta protein observed biochemically (Fig. 8C).

In addition to increasing peak current density, co-expression of alpha with beta increased the number of patched cells expressing detectable Ca currents between 24-60 h post-transfection. Only 3-5% of alpha cells exhibited low density Ca currents, while 50-80% of alpha/beta cells exhibited high density Ca currents. These data are consistent with the finding that co-expression of the beta subunit is necessary for the acquisition of high affinity DHP binding and membrane immunostaining of the alpha subunit.


DISCUSSION

The combined biochemical, immunochemical, and electrophysiological analyses described here revealed heretofore unappreciated characteristics of the beta subunit of Ca channels and subunit interactions in mammalian cells. Overall, the results argue that the beta subunit acts to increase and/or stabilize the population of functional channel complexes in the membrane in addition to modulating properties of channel conduction. From previous studies in Xenopus oocytes(3, 4, 5, 6, 7, 8) and mammalian cells(9) , it is clear that beta subunits can enhance peak current amplitudes and modulate aspects of activation and inactivation. In addition, in mammalian cells, beta subunits have been shown to enhance DHP or -conotoxin binding to either L-type or N-type alpha(1) subunits, respectively(5, 8, 9, 28, 29, 30, 31) . Additionally, recent studies have identified sites that are involved in direct interactions between the alpha(1) and beta subunits(32, 33, 34) . While these results collectively support a model of a multimeric channel complex whose properties are modulated by the beta subunits, little has been reported about the biochemical events that may be involved in these processes.

An unexpected finding here was that the beta subunit itself appears to behave as a membrane protein. Since none of the cloned beta subunit cDNAs predict that any of these proteins have transmembrane-spanning domains, and since the proteins are predicted to be extremely hydrophilic, it might have been expected that the beta subunit would be cytosolic in the absence of other channel subunits. However, in the present studies we found no evidence for the beta subunit in ``cytosolic'' fractions (supernatants after high speed centrifugation), and the immunocytochemistry clearly demonstrated staining of the beta subunit in the membrane periphery. Conceivably the beta subunit associates with other membrane proteins or with cytoskeletal proteins, to become membrane localized. This hypothesis is consistent with the relatively poor solubility of beta in Triton X-100, since insolubility in this detergent has long been a characteristic of cytoskeletally associated proteins. A cyoskeletal localization for the beta subunits is attractive in view of the potential role of the cytoskeleton in the inactivation and ``clustering'' of Ca channels(35, 36, 37) . We considered the possibility that the membrane localization of the beta subunit might be conferred via an association with other Ca channel subunits that might be endogenously expressed in HEK cells. However, this seems unlikely since Ca channel proteins are typically of very low density and it is unlikely that the HEK cells would express sufficient amounts of endogenous Ca channel subunits to allow for the substantial immunostaining we observed with the relatively ``overexpressed'' beta subunit at the periphery. In support of this view, electrophysiological studies have revealed that HEK cells possess very low or undetectable endogenous Ca currents (data not shown; 9, 30).

Another unexpected finding was that the beta subunit was expressed as multiple forms, due to unidentified post-translational modifications. It is unclear whether the multiple isoforms represent sequential modifications of the nascent 68-kDa protein, or whether these isoforms are differential modifications of the 68-kDa protein. The differential solubility of beta in salt and detergent suggests that a post-translational modification might lead to tighter association of some forms of beta with the membrane. Of particular interest was that the co-expression of the beta subunit promoted the appearance of the alpha(1) subunit at the plasma membrane. This correlated with the acquisition of high affinity DHP binding and appearance of large Ca currents. In the absence of the beta subunit, the alpha(1) subunit was largely intracellular and no saturable ligand binding could be quantified. Corresponding Ca currents were of a low density and could only be observed in a very few percentage of cells. In contrast, in alpha/beta cells, alpha(1) localized to the membrane, resulting in saturable ligand binding as well as a higher percentage of patched cells exhibiting high density calcium currents. The data from the biochemical and electrophysiological analyses are consistent with the hypothesis that the production of large numbers of functional channels is limited by the beta subunit copy number. The interaction with the beta subunit could help target the alpha(1) subunit to the membrane, and/or stabilize the alpha(1) subunit, thus increasing the probability of membrane insertion. With regard to the latter possibility, while the half-life of the alpha subunit was not altered by the beta subunit, we were unable to measure whether the half-life of the alpha(1) subunit in the plasma membrane versus that in intracellular compartments might be different. The oligomerization of the alpha and beta subunits may occur co-translationally, facilitating the insertion of functional channel complexes into the membrane and/or the formation, stabilization, and membrane targeting of channel complexes.

Our conclusion that the beta subunit increases the amount of alpha subunit at the plasma membrane is in contrast to findings in Xenopus oocytes, where analysis of charge movement was used to determine that co-expression of the beta subunit did not result in increased numbers of channels at the cell surface(11) . In addition, recent studies have demonstrated that the beta subunit did not cause increased immunoreactive alpha in oocyte plasma membranes(40) . However, Marban and colleagues have recently found that in transiently transfected HEK cells, co-expression of the beta subunit increases the number of alpha channels at the membrane as measured by charge movement(^5); this observation is consistent with the results of this paper. It is likely that differences between Xenopus and mammalian cell expression systems may account for the differences in results obtained regarding the effect of beta in these systems.

The present results and those of others (38) with regard to an inability to detect saturable DHP binding to cells expressing the alpha-subunit alone differs somewhat from previous results which reported that the expressed alpha subunit alone bound DHPs with high affinity; however, the level of binding reported was at the lower limit of detection of specific binding with this ligand(5, 8, 9, 28, 31, 39) . Notably, others demonstrated a very rapid dissociation of ligand from the alpha subunit in the absence of the beta subunit when expressed in COS cells(38) . This effect appeared to explain the lack of saturable ligand binding that was observed (38) and may also explain the results obtained in this study, as well as the very low levels of DHP binding reported from previous studies(28, 31, 39) . The increased targeting of alpha to the membrane by the beta subunit provides an explanation for the increase in functional channel at the plasma membrane, as measured by whole-cell currents and intact-cell ligand binding, in the absence of any increase in alpha protein levels.

In summary, the results described here suggest multiple roles for the beta subunit in the processing and functioning of the L-type channel. In addition, further studies on the functional effects of co-expressing the alpha and beta subunits in transiently transfected HEK cells have revealed diverse effects on peak current amplitude and Ca-dependent inactivation which are dependent upon levels of beta expression. (^6)The new information presented here argues that the beta subunit can localize to the membrane alone and that interactions between alpha and beta occur prior to channel targeting to the plasma membrane. These interactions appear to be critical for the proper transport and/or targeting of oligomerized subunits to the plasma membrane, and may also be important for conformational stabilization of the channel complex.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL23306 (to M. M. H.) and AR43113 (to E. R.), and by grants from the American Heart Association (to E. R.) and the American Heart Association of Metropolitan Chicago (to R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Research Service Award Predoctoral Training Grant 5 T32 ES07124 in the Environmental Sciences.

Permanent address: A. A. Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev 252024, Ukraine.

**
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; mhosey@nwu.edu.

(^1)
The abbreviations used are: HEK, human embryonic kidney; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; DHP, dihydropyridine.

(^2)
T. S. Puri and M. M. Hosey, manuscript in preparation.

(^3)
X. Zhao, B. Gerhardstein, and M. M. Hosey, unpublished observations.

(^4)
T. Y. Gao and M. M. Hosey, unpublished observations.

(^5)
T. J Kamp and E. Marban, personal communication.

(^6)
R. E. Shirokov, G. Ferreira, J. Yi, J. Zhou, A. J. Chien, M. M. Hosey, and E. Rios, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Rebecca Brawley Terns for assistance in the development and production of the Card C antibody. We also thank Dr. Robert Decker for generous use of fluorescence microscope and for helpful suggestions and technical advice.


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