AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 288: H2363-H2374, 2005. First published December 22, 2004; doi:10.1152/ajpheart.00348.2004
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Unique modulation of L-type Ca2+ channels by short auxiliary {beta}1d subunit present in cardiac muscle

Risa M. Cohen,2,* Jason D. Foell,1,* Ravi C. Balijepalli,1 Vaibhavi Shah,3 Johannes W. Hell,3 and Timothy J. Kamp1,2

Departments of 1Medicine and 2Physiology, University of Wisconsin, Madison, Wisconsin; and 3Department of Pharmacology, University of Iowa, Iowa City, Iowa

Submitted 12 April 2004 ; accepted in final form 21 December 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies have identified a growing diversity of splice variants of auxiliary Ca2+ channel Cav{beta} subunits. The Cav{beta}1d isoform encodes a putative protein composed of the amino-terminal half of the full-length Cav{beta}1 isoform and thus lacks the known high-affinity binding site that recognizes the Ca2+ channel {alpha}1-subunit, the {alpha}-binding pocket. The present study investigated whether the Cav{beta}1d subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Cav{beta} subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Cav{beta}1d identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Cav{beta}1b subunit promoted membrane trafficking of the pore-forming {alpha}1C (Cav1.2)-subunit to the surface membrane, whereas the Cav{beta}1d subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Cav{beta}1d with the {alpha}1C-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Cav{beta}1b coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Cav{beta}1b or Cav{beta}1d significantly increased mean open probability four- to fivefold relative to the {alpha}1C-channels alone, but only Cav{beta}1b coexpression increased the number of channels observed per patch. In conclusion, the Cav{beta}1d isoform is expressed in heart and can modulate the gating of L-type Ca2+ channels, but it does not promote membrane trafficking of the channel complex.

electrophysiology; ion; heart; splice variant

CALCIUM CHANNELS ARE FOUND IN many excitable tissues and are intricately involved in important cellular processes including genesis of action potentials, excitation-contraction coupling, and gene expression. Voltage-dependent Ca2+ channels are multisubunit proteins that consist of a pore-forming {alpha}1-subunit that is associated with auxiliary subunits including the cytoplasmic {beta}-subunit, a membrane-associated {alpha}2{delta}-subunit, and a {gamma}-subunit (10). The ultimate functional properties of Ca2+ channels are determined not only by the pore-forming {alpha}1-subunits but also by the combination of auxiliary subunits that finely tune the channel properties.

The auxiliary Ca2+ channel Cav{beta} subunit is encoded by four different genes with a rich array of alternatively spliced isoforms that are expressed in a tissue- and species-specific pattern (6). For example, in canine and human ventricles, we have previously found (20) evidence for a total of 18 different Cav{beta} isoforms. The genes typically encode proteins of 400–600 amino acids in length and are composed of five general domains (Fig. 1). Domains 2 and 4 are widely conserved across all four Cav{beta} genes and contain Src homology-3 (SH3) and guanylate kinase (GK) motifs, respectively. Recent crystallographic studies (11, 35, 45) have demonstrated that Cav{beta} subunits are members of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which are characterized by closely interacting SH3 and GK domains. High-affinity binding of Cav{beta} subunits to the {alpha}1-subunit occurs in a hydrophobic groove in the Cav{beta} subunit GK domain that is referred to as the {alpha}-binding pocket (ABP). All full-length Cav{beta} subunits include a similar core formed by the interacting SH3 and GK domains including the ABP, and variability among the different Cav{beta} isoforms occurs primarily in domains 1, 3, and 5. In addition, recent studies have also identified short or truncated Cav{beta} isoforms that result from alternative splicing and a frame shift that causes an early stop codon (20, 25, 26). These short or "d" isoforms are truncated before domain 4, and the resulting proteins are approximately one-half the size as the full-length isoforms and thus lack the GK domain with its high-affinity binding site for the {alpha}1-subunit.



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  		Fig. 1. Structure of human Cav{beta}1-splice variants and constructs studied. Conserved domains among the four Cav{beta} subunit genes; gray regions) and variable domains (open bars) are shown. Unique seven amino acids found only in Cav{beta}1d and not other Cav{beta}1-splice variants in domain (D) 3 owing to alternative splicing and a frame shift are indicated (cross-hatched areas). Cav{beta}1d{Delta}1–13 and Cav{beta}1b{Delta}1–13 lack the first 13 amino acids of domain 1.

 
The functional roles of the full-length Cav{beta} subunits have been extensively explored using heterologous expression studies. Cav{beta} subunits play critical roles in membrane trafficking of the channel complex and in regulation of voltage-dependent gating (4, 13, 17, 22, 29, 30). The Cav{beta} subunit binds to the endoplasmic reticulum (ER) retention signal in the I-II loop of the {alpha}1-subunit, which allows channels to traffic to the surface membrane (4). Furthermore, Cav{beta} subunits not only allow for membrane trafficking of the channel complex; they also can play a role in determining the subcellular localization of channels on the surface membrane (7, 15, 20). Cav{beta} subunits have been demonstrated to differentially regulate activation, inactivation, recovery from inactivation, and voltage-dependent facilitation of heterologously expressed channels (18, 28–30, 40). Modulation of voltage-dependent gating can be finely regulated by different splice variants from the same Cav{beta} subunit gene. For example, splice variants of Cav{beta}2 and Cav{beta}4 genes, which differ only in the proximal NH2 terminus (domain 1), have distinct effects on channel properties including steady-state activation and voltage-dependent inactivation gating (20, 24, 43).

The shorter splice forms or "d" isoforms of the Cav{beta} genes have only recently been identified, and their functional properties have not yet been completely characterized. The short isoforms lack the ABP, so their effects on expressed {alpha}1-channels may be quite different or absent compared with full-length Cav{beta} isoforms. All four Cav{beta} genes can encode short Cav{beta} subunits using similar alternative splicing (20, 25, 26), but only the Cav{beta}3 and Cav{beta}4 short isoforms have been functionally characterized. In the case of the Cav{beta}4 gene, a short isoform was shown to be involved in gene regulation and only caused minor changes in voltage-dependent gating (25). The short form of the Cav{beta}3 isoform did not clearly alter whole cell currents, but at the single-channel level effects on gating were observed (26). No studies have yet proven that the short isoforms are expressed at the protein level in native tissues, including heart. In addition, no investigations have directly addressed whether the short Cav{beta} subunits promote membrane trafficking of the channel complex compared with full-length Cav{beta} subunits. Last, no functional characterization of a heterologously expressed short-splice variant from the Cav{beta}1 gene has been reported.

The purpose of the present work was to determine whether the short Cav{beta}1 splice variant Cav{beta}1d is expressed at the protein level in heart and to determine its functional effects on membrane trafficking and voltage-dependent gating of {alpha}1C-encoded L-type Ca2+ channels. A preliminary account of these studies has been reported (14).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Heterologous expression of Ca2+ channel subunits in HEK-293 cells. HEK-293 cells were cultured in DMEM supplemented with 10% fetal calf serum (Harlan; Indianapolis, IN), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The HEK-293 cells were grown on 15-mm glass coverslips coated with type I collagen from rat tails (Sigma; St. Louis, MO) and were transiently transfected with the subunit(s) of interest using the calcium phosphate method (Invitrogen; Carlsbad, CA). The rabbit full-length {alpha}1C (Cav1.2)-subunit (33), except for alternative splicing in domain IV S3 (41), subcloned into pGW1H (British Biotechnology; Oxford, UK), was expressed alone or in a 1:1 molar ratio with human {beta}1b- (GenBank accession no. M92303) or human {beta}1d- (GenBank accession no. AY393857) subunit subcloned into pcDNA3.1. The channel subunits were cotransfected with pSV40Tag to increase expression levels and green fluorescent protein (GFP)pRK5 to identify transfected cells (36). Cells were then used for whole cell electrophysiology or imaging experiments the day after transfection.

{beta}1d-Antibody and Western blot analyses. A polyclonal rabbit anti-Cav{beta}1d antibody was produced using an epitope specific to the unique COOH terminus of the Cav{beta}1d subunit (KPASDRACAPL; the NH2-terminal lysine is not part of the original Cav{beta}1d sequence but was added to aid in coupling to the carrier). The Cav{beta}1d COOH-terminal peptide was synthesized, purified, coupled to BSA with glutaraldehyde, and used to immunize rabbits as previously described (50). The resulting antibody was affinity purified on the peptide used for immunization coupled to CNBr-activated Sepharose 4B.

Sarcolemmal-, T-tubular-, and dyadic-enriched membrane fractions (FI, FII, and FIII, respectively) were prepared from canine left ventricular tissue using differential and discontinuous sucrose density-gradient centrifugation (1). Proteins (60 µg) from the isolated canine ventricular membrane fractions and transfected HEK-293 cells were solubulized in sample buffer (that contained 62.5 mM Tris·HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.1% bromophenol blue) by warming to 60°C for 30 min (23) and were separated by SDS-PAGE using 12% bis-acrylamide gels as described by Laemmli (31). After separation, proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories; Hercules, CA) by blotting for 1 h at 105 V. Nonspecific binding sites were blocked by immersion of membranes for 2 h at room temperature in Tris-buffered saline detergent (0.1% Tween-20) that contained 5% (wt/vol) dried skim milk. Membranes were then probed with rabbit polyclonal anti-Cav{beta}1d primary antibody (1:500 dilution). Goat anti-rabbit immunoglobulin linked to horseradish peroxidase (1:50,000 dilution) detected bound primary antibody. Immunoreactivity was visualized using the ECL peroxidase-based chemiluminescent detection system (Amersham Life Sciences; Cleveland, OH).

Immunofluorescence confocal imaging. Immunolabeling was performed on transiently transfected HEK-293 cells using a rabbit polyclonal antibody directed against {alpha}1C-protein (recognizing a region in the central cytosolic loop between {alpha}1C-domains II and III; Ref. 16). Transfected HEK-293 cells grown overnight were initially fixed with 2% paraformaldehyde in PBS for 10 min. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and then quenched for aldehyde groups in 0.75% glycine in PBS for 10 min. After cells were washed with PBS for 2–5 min, they were incubated with 1 ml of blocking solution (that contained 2% BSA, 5% goat serum, and 0.05% NaN3 in TBS) for 1 h to block nonspecific binding. Subsequently, the cells were incubated with the polyclonal anti-{alpha}1C-antibody (a 1:500 dilution) for 2 h in blocking solution at room temperature. Excess primary antibody was washed off with blocking solution (for 3–10 min). Cells were then incubated with Alexa Fluor 568 goat anti-rabbit IgG antibody (a 1:500 dilution, 2 mg/ml for 2 h; Molecular Probes; Eugene, OR), subsequently washed with blocking solution (for 3–10 min), and mounted onto a slide in a 50:50 glycerol-PBS solution. To identify nonspecific binding, control experiments with secondary antibody alone and experiments in mock transfected cells were performed.

Imaging was performed with an MRC 1024 laser scanning confocal microscope equipped with a mixed-gas (Ar-Kr) laser operated by 24-bit LaserSharp software (Bio-Rad). The Bio-Rad system was mounted on a Nikon Diaphot 200 inverted microscope. Acquisition in the red channel utilized excitation at a 568-nm wavelength with emission detected at 605 ± 16 nm. Cells were randomly selected and used for image analysis. Images were scanned using a x60 objective.

Biotinylation of cell surface protein and immunoblotting. HEK-293 cells were grown in 100-mm culture dishes and individually transfected with the subunit(s) of interest, and the rabbit full-length {alpha}1C-subunit was expressed alone or in a 1:1 molar ratio with human {beta}1b- or {beta}1d-subunits. After 48 h, cells were washed three times with PBS at room temperature (22–25°C), and cell surface proteins were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit [0.5 mg/ml sulfo-NHS-(LC)-biotin; Pierce; Rockford, IL] in PBS. After incubation at 4°C for 1 h, cells were washed five times with ice-cold PBS to remove any remaining biotinylation reagent. Cells were then harvested in lysis buffer (that contained 130 mM NaCl, 50 mM Tris·HCl, 2 mM EGTA, 1% Nonidet P-40, and 1% Triton X-100) and protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 50 µg/ml benzamidine, 50 µg/ml leupeptin, and 5 µM pepstatin A; Sigma). Lysate proteins were quantified with a bicinchoninic acid assay (Pierce). Proteins (150 µg/reaction) were mixed with anti-{alpha}1C-antibody (10 µg) and incubated overnight at 4°C, and then 50 µl of a 1:1 slurry of protein A-Sepharose beads was added to the incubation mix for an additional 1 h at 4°C. Beads were pelleted, washed thoroughly in lysis buffer, and incubated in Laemmli sample buffer at 60°C for 30 min. Bound proteins were separated using SDS-PAGE, which was followed by Western blot analysis as described earlier using either rabbit anti-{alpha}1C-primary antibody at a 1:200 dilution or goat anti-biotin antibody (Abcam; Cambridge, MA) at a 1:20,000 dilution.

Whole cell electrophysiology. Coverslips with HEK-293 cells were placed in the bottom of the perfusion chamber (Warner Instruments; Hamden, CT), and transfected cells that expressed green fluorescent protein were detected by epifluorescence microscopy. Experiments were performed at 22–24°C. The ruptured whole cell pipette solution consisted of (in mM) 114 CsCl, 10 EGTA, 10 HEPES, and 5 MgATP (pH 7.2 with CsOH). Cells were initially bathed in Ca2+-Tyrode solution that contained (in mM) 1.8 CaCl2, 142 NaCl, 5.4 KCl, 1.0 MgCl2, 0.33 Na2H-PO4, and 5 HEPES (pH 7.40 with NaOH). After a gigaseal was formed and the whole cell ruptured configuration was obtained, the cells were bathed in a solution that consisted of (in mM) 10 BaCl2, 133 CsCl, and 10 HEPES (pH 7.4 with CsOH). For perforated patch experiments, the pipette solution consisted of 100 mM Cs-glutamine, 0.5 mM CaCl2, 40 CsCl, 10 HEPES, and 60 ng of amphotericin B (pH 7.2 with CsOH). The bath solution was the same as that used for the ruptured patch configuration. The Ca2+ channel activator SDZ+202-791 (1 µM; Biomol Research Laboratories; Plymouth Meeting, PA) in bath solution was tested in some experiments. Whole cell currents were recorded using an Axopatch 200B amplifier (Axon Instruments; Foster City, CA). Data were sampled at 25 kHz and filtered at 5 kHz. The holding potential was –80 mV. Liquid junction potential was determined to be –3.4 mV under these conditions, and data were not corrected for this offset. Electrodes were pulled using a micropipette puller (Sutter Instruments; Novato, CA) and heat polished to obtain resistances of between 1.5 and 3.5 M{Omega} when filled with internal solution. Data analysis was performed using pClamp (Axon Instruments) and Origin (Microcal; Northampton, MA) software. Whole cell conductance (G) was calculated from peak current divided by the driving force, which is determined by the test potential minus the reversal potential. The reversal potential used for these calculations was +60 mV. The whole cell conductance-voltage (G-V) curve was plotted and fit by a Boltzmann function using the equation

where Gmax is the maximum whole cell conductance, V is the potential, V1/2 is the potential at which half-activation occurs, and k is the slope value.

Single-channel electrophysiology. Single-channel data were collected using the cell-attached patch configuration with an Axopatch 200B amplifier. Borosilicate glass electrodes were pulled and fire polished to resistances of between 2 and 6 M{Omega} when filled with internal solution. Electrodes were also coated with a silicone elastomer for additional noise reduction (Sylgard; Dow Corning; Midland, MI). The bath solution consisted of (in mM) 110 potassium aspartate, 30 KCl, 3.8 MgCl2, 5 HEPES, 5 EGTA, 1.2 CaCl2, and 10 dextrose. The Ca2+ channel activator SDZ+202-791 (1 µM) was included in the bath solution. The pipette solution was composed of (in mM) 100 BaCl2, 20 tetraethylammonium chloride, and 10 HEPES. Junction potential was calculated to be approximately –9.3 mV for these solutions, and test potentials were corrected accordingly. Data were sampled at 10 kHz and filtered at 1–2 kHz. Data were analyzed with a custom program that used Microcal Origin. To determine mean open probability (Po), the data were converted into idealized events using traces corrected for capacity transients and leak current by subtracting the averages of traces without openings. Channel-open events were identified by a half-height criterion.

Statistics. All values are presented as means ± SE. Statistical comparisons were made by use of the two-tailed Student's t-test. Differences with P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of {beta}1d-protein in heart. Our previous study identified the mRNA encoding Cav{beta}1d protein in canine and human hearts (20), but the presence of the encoded protein in heart was not determined. To probe for Cav{beta}1d protein expression, we produced a specific rabbit polyclonal antibody directed against the unique carboxyl terminus of Cav{beta}1d that results from the alternative splicing and frame shift in this isoform. Western blot analysis of membranes from HEK-293 cells transfected with Cav{beta}1a, Cav{beta}1b, or Cav{beta}1d demonstrated that the Cav{beta}1d antibody recognized a 23-kDa protein only in the Cav{beta}1d-transfected cells of the predicted molecular mass of 23 kDa (Fig. 2). The specificity of this antibody was highlighted by the fact that no significant immunoreactivity was found in the membranes from Cav{beta}1a- or Cav{beta}1b-transfected cells despite the amino acid sequence of Cav{beta}1d protein being identical to the first six exons of Cav{beta}1a and Cav{beta}1b (except for the final seven amino acids, which are found uniquely in Cav{beta}1d). Next, the presence of Cav{beta}1d protein in cardiac muscle was tested using Western blot analysis of canine and human ventricular membranes. Canine left ventricular membranes were prepared using a discontinuous sucrose gradient that isolated membrane fractions enriched in the surface sarcolemma (F1), T-tubular sarcolemma (F2), and junctional complexes (F3). The Cav{beta}1d antibody readily detected a 23-kDa protein in all three canine ventricular membrane fractions that was enriched in the membrane fractions relative to crude homogenate (band seen with longer exposure; data not shown). Likewise, a 23-kDa band was detected in enriched membranes (pooled F1, F2, and F3) from human ventricle. These results demonstrate the expression of Cav{beta}1d subunit at the protein level in canine and human hearts.



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  		Fig. 2. Specific detection of Cav{beta}1d protein in canine heart, human heart, and transfected HEK-293 cells. Western blot analysis was performed using anti-Cav{beta}1d antibody on canine membrane fractions (F) enriched in surface sarcolemma (F1), T-tubular sarcolemma (F2), junctional complexes (F3), and crude membrane homogenate (H). A sample of enriched membranes from human ventricle was also tested (middle lane). Membrane homogenates from HEK-293 cells transfected with Cav{beta}1a, Cav{beta}1b, or Cav{beta}1d were evaluated (right three lanes). Amount of membrane protein loaded in each lane was 60 µg.

 
Membrane trafficking of {alpha}1C-channels by {beta}1b- and {beta}1d-subunits. An important property of full-length Cav{beta} subunits is to promote membrane trafficking of high-voltage activated Ca2+ channel {alpha}1-subunits to the surface membrane (4, 13, 27, 29). The {beta}-interaction domain (BID) region in domain 4 of the Cav{beta} subunit has been suggested (4) to be essential to alleviate ER retention of the {alpha}1-subunits, and more recent structural experiments have identified the ABP that lies distal to the BID in domain 4 as the region of the Cav{beta} that binds to the {alpha}-interaction domain (AID) of {alpha}1-subunits. Therefore, we predicted that a fundamental difference between short and full-length Cav{beta} subunits would be the ability to chaperone {alpha}1-subunits to the surface membrane. HEK-293 cells were transfected with {alpha}1C-subunit alone or in combination with a representative full-length Cav{beta}1 subunit, Cav{beta}1b, or the short Cav{beta}1d. Immunolabeling was performed on transiently transfected HEK-293 cells using a rabbit polyclonal antibody to {alpha}1C-subunit (16). Confocal microscopy was used to image immunolabeled cells that expressed {alpha}1C-subunits alone (Fig. 3, A and B), {alpha}1C + Cav{beta}1b subunits (Fig. 3, C and D), and {alpha}1C + Cav{beta}1d subunits (Fig. 3, E and F). Comparison of the phase-contrast images (Fig. 3, left) with the fluorescence images (Fig. 3, right) of the cells demonstrates that similar immunolabeling of {alpha}1C-subunits was observed for {alpha}1C-subunits alone and {alpha}1C + Cav{beta}1d-expressing cells with predominately an intracellular reticulate pattern with strong perinuclear labeling consistent with localization to the ER-Golgi system. Careful inspection of Fig. 3D demonstrates that {alpha}1C-protein coexpressed with Cav{beta}1b again shows intracellular immunofluorescence, but there is also clear membrane localization of {alpha}1C-subunits as indicated by the arrows. The specificity of the immunofluorescence is demonstrated by the minimal fluorescence in a third cell (Fig. 3, A and B) that was not transfected. These results suggest that Cav{beta}1d does not effectively promote membrane trafficking of {alpha}1C-subunits to the surface membrane in contrast with full-length Cav{beta}1b.



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  		Fig. 3. Cav{beta}1b but not Cav{beta}1d promotes membrane trafficking of {alpha}1C-subunits to surface membrane. Confocal images of HEK-293 cells transfected with {alpha}1c-subunits alone (A and B), {alpha}1c + {beta}1b (C and D), and {alpha}1C + {beta}1d (E and F) subunits immunolabeled with an {alpha}1C-antibody are shown. Phase-contrast images (left) of the cells and matched immunofluorescence using an Alexa Fluor 568 anti-rabbit secondary antibody (right) are shown. Cell surface membranes were determined by phase-contrast confocal images (arrows).

 
To confirm the immunocytochemistry experiments, biotinylation experiments were performed in intact cells to detect surface membrane {alpha}1C-channels in the absence and presence of Cav{beta}-subunit coexpression. Intact HEK-293 cells expressing {alpha}1C-subunit alone, {alpha}1C + Cav{beta}1b, and {alpha}1C + Cav{beta}1d and mock transfected cells were surface biotinylated, and the whole cell lysates were immunoprecipitated using anti-{alpha}1C-antibody and subsequent Western blot analysis of the immunoprecipitates. A representative Western blot (Fig. 4A) probed with anti-biotin antibody demonstrates a band at 240 kDa seen most prominently with Cav{beta}1b coexpression relative to {alpha}1C-subunits alone or {alpha}1C + Cav{beta}1d. When the blot was stripped and reprobed with anti-{alpha}1C-antibody, the same 240-kDa protein was detected, which demonstrates that the biotinylated protein was the {alpha}1C-subunit. The greater signal on the anti-biotin blot for the {alpha}1C + Cav{beta}1b lane relative to {alpha}1C and {alpha}1C + Cav{beta}1d suggests that Cav{beta}1b-subunit coexpression leads to the greatest surface membrane expression of {alpha}1C-subunits. Interestingly, the anti-{alpha}1C-antibody blot showed the greatest signal in the {alpha}1C + Cav{beta}1b lane; it showed a greater abundance of {alpha}1C-subunits in the immunoprecipitates despite identical transfection and an identical protein amount loaded onto gels. Therefore, coexpression of Cav{beta}1b may also stabilize the heterologously expressed {alpha}1C-subunit and lead to a greater steady-state amount. In addition, other proteins were recognized with the anti-biotin blots that are associated with {alpha}1C-subunits, but their identities are not known. The specificity of the findings is demonstrated by lanes for mock-transfected cells that showed no signal for anti-biotin or anti-{alpha}1C-antibodies. These results were typical of five experiments. Overall, the biotinylation experiments demonstrate a greater expression of surface membrane {alpha}1C-subunits when Cav{beta}1b is coexpressed compared with Cav{beta}1d coexpression or {alpha}1C-subunit expression alone.



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  		Fig. 4. Surface labeling of {alpha}1C-channels through biotinylation. Cell surface biotinylation of {alpha}1C-channels was examined using HEK-293 cells transiently transfected with {alpha}1C-, {alpha}1C + Cav{beta}1b, or {alpha}1C + Cav{beta}1d subunits. Biotin-labeled HEK-293 cell lysate was immunoprecipitated with anti-{alpha}1C-antibody and subjected to Western blot analysis with anti-biotin antibody (A) then stripped and reprobed with anti-{alpha}1C-antibody (B); the 240-kDa {alpha}1C-subunit is indicated.

 
Effects of {beta}1b- and {beta}1d-subunits on whole cell electrophysiology of {alpha}1C-channels. To directly compare the functional modulation of {alpha}1C-channels by the full-length Cav{beta}1b to the short Cav{beta}1d subunits, transfected HEK-293 cells were studied using the whole cell configuration of the patch-clamp technique. From a holding potential of –80 mV, a series of 50-ms depolarizing pulses was applied from –50 to +80 mV in 10-mV steps, and representative raw current traces at –30, –10, and +10 mV are shown in Fig. 4A for all subunit combinations. Expression of the Ca2+ channel {alpha}1C-subunit alone in HEK-293 cells resulted in small inward currents carried by 10 mM Ba2+ that were typical of our previous studies and those of others using this expression system (21, 29, 36). The {alpha}1C-currents activate in a voltage-dependent fashion, and the resulting current-voltage (I-V) curve (Fig. 5B) is bell shaped, which is characteristic of L-type Ca2+ channels. As anticipated, coexpression of the full-length Cav{beta}1b with {alpha}1C-subunits resulted in markedly greater current densities that were ~15-fold larger at the peak of the I-V curve compared with {alpha}1C-subunits alone (coexpression, –96.4 ± 14.2, n = 11; {alpha}1C alone, –6.4 ± 1.2 pA/pF, n = 6; Fig. 5). In contrast with the striking effect of Cav{beta}1b on current density, coexpression of Cav{beta}1d with {alpha}1C-subunits did not result in a significant change in the measured currents compared with expression of {alpha}1C-subunits alone (current at peak of I-V curve for {alpha}1C + Cav{beta}1d, –6.69 ± 1.02 pA/pF, n = 14).



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  		Fig. 5. Effects of Cav{beta}1b and Cav{beta}1d coexpression on {alpha}1C-whole cell Ba2+ current (IBa) in transiently transfected HEK-293 cells. Whole cell IBa was measured from HEK-293 cells transfected with {alpha}1C-subunits alone, {alpha}1C + {beta}1b, or {alpha}1C + {beta}1d subunits. Cells were held at –80 mV and given a series of 50-ms pulses from –50 to +80 mV. Representative raw current traces (test potentials of –30, –10, and +10 mV) are shown (A). Average IBa-voltage (V) data were normalized to cell capacitance (B). Normalized whole cell conductance (G/Gmax) is plotted vs. voltage (C). Average data were fit to Boltzmann distributions.

 
Closer inspection of the I-V curves suggested that coexpression of Cav{beta}1b resulted in a hyperpolarizing shift in the voltage dependence of channel activation that was comparable to previous studies with this subunit and many other auxiliary Cav{beta} subunits (8, 9, 17, 21, 37). To more directly assess the impact of Cav{beta}-subunit coexpression on the voltage dependence of current activation, we plotted G calculated from peak current vs. test potential (V; see MATERIALS AND METHODS). Normalized G-V curves are shown for {alpha}1C-, {alpha}1C + {beta}1b-, and {alpha}1C + {beta}1d-subunit combinations in Fig. 5C, and the data were fit to Boltzmann distributions that resulted in V1/2 values of –3.9 ± 0.8, –10.2 ± 1.7, and –5.0 ± 1.1 mV, respectively; and for k values of 7.4 ± 0.6, 4.2 ± 1.7, and 6.6 ± 0.9 mV, respectively. These data demonstrate that coexpression of Cav{beta}1b with the {alpha}1C-subunit results in a 6.3-mV hyperpolarizing shift in the V1/2 for current activation with a steeper activation curve, but coexpression of Cav{beta}1d has no significant effect on the voltage dependence of current activation.

Another prominent and well-characterized effect of Cav{beta} subunits is to modulate the kinetics of current decay or inactivation (12, 34, 36, 49). From a holding potential of –80 mV, 250-ms voltage steps were given to –20, –10, 0, 10, and 20 mV. Current inactivation was measured as the fraction of current remaining at 50 ms (r50) and 200 ms (r200) relative to the peak current. Representative normalized current traces at +10 mV for each subunit combination are plotted in Fig. 6A, and average data over the range of membrane potentials for r50 and r200 are shown in Fig. 6, B and C. There was no significant difference between {alpha}1C-subunits alone and {alpha}1C + Cav{beta}1d in current decay at any potential measured for r50 and r200. However, coexpression of Cav{beta}1b significantly accelerated current decay at positive potentials as can be seen in both the r50 and r200 measurements.



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  		Fig. 6. Cav{beta}1b but not Cav{beta}1d increases rate of IBa inactivation. IBa decay at +10 mV was compared for cells expressing {alpha}1C-subunits alone, {alpha}1C + {beta}1b, and {alpha}1d + {beta}1b subunits. Representative normalized raw current traces at +10 mV are plotted (A). Average data comparing ratio of IBa remaining at 50 ms (r50; B) or 200 ms (r200; C) relative to peak IBa for different Ca2+ channel subunit combinations (n = 5, 10, and 10 for {alpha}1C-subunits alone, {alpha}1C + {beta}1b, and {alpha}1C + {beta}1d, respectively; *P < 0.05 relative to {alpha}1C-subunits only).

 
Cav{beta}1b and Cav{beta}1d subunits modulate single-channel properties of {alpha}1C-channels. Our experiments to this point demonstrate that Cav{beta}1d is expressed as a protein in transfected HEK-293 cells, but no effects were detected on the whole cell currents with Cav{beta}1d expression, which is in sharp contrast to the marked effects of Cav{beta}1b coexpression. We decided to examine single-channel currents in the transfected HEK-293 cells, because Hullin et al. (26) recently showed that there was no measurable effect of another short Cav{beta} subunit, Cav{beta}3d, when it was coexpressed with {alpha}1C-subunits in whole cell recordings, but modulation was revealed by single-channel studies.

Transiently transfected HEK-293 cells were studied with the cell-attached patch-clamp technique to measure single-channel currents. Cells were transfected with the {alpha}1C-subunit with or without auxiliary Cav{beta}1 subunits. In these experiments, truncated amino-terminal subunits (deletion of first 13 amino acids), Cav{beta}1b{Delta}1-13 and Cav{beta}1d{Delta}1-13, were studied in an effort to begin determination of the essential amino acids for modulation of the {alpha}1C-currents. Whole cell studies of expressed Cav{beta}1b{Delta}1-13 and Cav{beta}1d{Delta}1-13 showed indistinguishable modulation of the whole cell currents relative to the nontruncated Cav{beta}1b- and Cav{beta}1d-subunits (data not shown). Single-channel studies were done in the presence of the Ca2+ channel activator SDZ+202-791 to stimulate single-channel activity and produce longer openings and thereby allow more accurate measurement of single-channel currents. Patches were held at –80 mV and given test pulses to –10 mV for 200 ms. Figure 7 shows representative current traces from multichannel patches with the different subunit combinations. Under our experimental conditions, patches containing only a single channel were never observed when Cav{beta} subunits were coexpressed. Inspection of the representative currents shows sparse openings with many null sweeps for the patches expressing {alpha}1C-subunits alone but greater channel activity in the patches coexpressing the auxiliary Cav{beta}1b{Delta}1-13 and Cav{beta}1d{Delta}1-13 subunits. Openings were typically long lived, which is as expected from the presence of SDZ+202-791. Ensemble average currents in Fig. 7B suggest that average currents were increased by coexpression of Cav{beta}1b or Cav{beta}1d subunits.



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  		Fig. 7. Cav{beta}1b and Cav{beta}1d increase measured single-channel activity when coexpressed with {alpha}1C-channels. HEK-293 cells transiently transfected with {alpha}1C-subunits alone, {alpha}1C + Cav{beta}1b, and {alpha}1C + Cav{beta}1d subunits were examined for single-channel activity using the cell-attached configuration of the patch-clamp technique with 1 µM SDZ+202-791 in the bath. Representative consecutive single-channel current traces for patches held at –80 mV that were given a 200-ms test pulse to –10 mV every 1 s are shown (A). Closed level (solid line), channel openings (downward deflections), and open levels (thin lines) are indicated. Ensemble average currents for each subunit combination are shown (B). Measured numbers of channels per patch were two for {alpha}1C alone, six for {alpha}1C + Cav{beta}1b, and seven for Cav{beta}1d.

 
In general terms, the apparent greater single-channel activity observed with Cav{beta}1-subunit coexpression could be due to changes in channel number in the patches and/or alterations in channel gating. Therefore, we sought to first measure channel number in each patch. By taking advantage of the presence of a Ca2+ channel activator and using a strong depolarization protocol to facilitate channel openings, we measured the number of stacked openings as an index of channel number as shown in Fig. 8A. As seen in Fig. 8B, the average number of channels detected per patch was significantly increased approximately eightfold by Cav{beta}1b{Delta}1-13 coexpression, whereas expression of Cav{beta}1d{Delta}1-13 did not significantly change the number of channels observed per patch compared with {alpha}1C-subunits alone.



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  		Fig. 8. Coexpression of Cav{beta}1b but not Cav{beta}1d increases the average number of {alpha}1C-channels present in cell-attached patches. Voltage protocol and five consecutive representative single-channel traces from a cell transfected with {alpha}1C- and Cav{beta}1b subunits are shown (A). To determine the number of channels present in a given patch, experiments were done in the presence of 1 µM SDZ+202-791 and with strong facilitating depolarizations to optimize our ability to detect stacked openings. Average numbers of channels per patch for each Ca2+ channel subunit combination are indicated (B).*P < 0.05, relative to {alpha}1C alone and {alpha}1C + {beta}1d.

 
Next we assessed the effect of Cav{beta} subunit coexpression on average single-channel gating by determining the mean Po for each subunit combination by correcting for the channel number. In Fig. 9A, mean Po is significantly increased by coexpression of either Cav{beta}1b{Delta}1-13 or Cav{beta}1d{Delta}1-13. Both subunits comparably increased Po in the range of fourfold. This observation provides the strongest evidence that the short Cav{beta}1d subunit can interact with the {alpha}1C-channel and modulate single-channel gating. Although coexpression of Cav{beta}1 subunits altered single-channel gating, they did not alter the single-channel conductance (g) of the expressed channels ({alpha}1C-subunits alone, 27.1 ± 0.1; {alpha}1C + Cav{beta}1d{Delta}1-13, 28.0 ± 0.1; and {alpha}1C + Cav{beta}1b{Delta}1-13, 26.3 ± 0.3 pS) as shown in Fig. 9B.



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  		Fig. 9. Comparison of mean open probability (Po) and single-channel conductance for {alpha}1C-channels alone or with Cav{beta}1b or Cav{beta}1d. Calculated mean Po values are shown for each Ca2+ channel subunit combination after correction for the number of channels present at a test potential of –10 mV (A). *P < 0.05 compared with {alpha}1C-subunits alone. Single-channel current is plotted as a function of test voltage for {alpha}1C-, {alpha}1C + Cav{beta}1b-, and {alpha}1C + Cav{beta}1d-expressing cells (B). Linear regression fit of {alpha}1C-subunit data is displayed. Single-channel conductance (g) was calculated from the slope of the fit lines for each subunit combination ({alpha}1C, 27.1 ± 0.1; {alpha}1C + Cav{beta}1b, 26.3 ± 0.03; and {alpha}1C + Cav{beta}1d, 28.0 ± 0.1 pS). There were no significant differences among groups for single-channel conductance.

 
To determine whether the similar single-channel gating behavior of Cav{beta}1b{Delta}1-13- and Cav{beta}1d{Delta}1-13-coexpressing channels was due to intrinsically similar modulatory properties of the subunits or was confounded by differential sensitivity to SDZ+202-791, we directly measured the effect of 1 µM SDZ+202-791. These experiments used the perforated patch technique to most closely simulate conditions for single-channel recordings. Both {alpha}1C + Cav{beta}1b{Delta}1-13- and {alpha}1C + Cav{beta}1d {Delta}1-13-transfected cells showed similar increases in current (90.3 ± 12.2%, n = 5 and 83.5 ± 5.0%, n = 4, respectively). Thus the similar single-channel gating changes imparted by Cav{beta}1b or Cav{beta}1d were not confounded by differences in sensitivity to SDZ+202-791.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we demonstrate expression of the recently identified Cav{beta}1d isoform as a 23-kDa protein in canine and human hearts, and we examine the functional properties of Cav{beta}1d using heterologous expression studies. In contrast with the full-length splice variants of the Cav{beta}1 gene, expression of Cav{beta}1d, which lacks the ABP, fails to traffic {alpha}1C-channels to the surface membrane. However, coexpression of the Cav{beta}1d subunit can modulate the gating properties of the expressed {alpha}1C-channels when examined at the single-channel level as manifest by a Cav{beta}1d subunit-induced increase in mean Po.

Lack of membrane trafficking by Cav{beta}1d subunit. The ability of Cav{beta} subunits to promote trafficking of Cav1.x and Cav2.x channels to the surface membrane has been demonstrated in a variety of studies using techniques ranging from intramembrane charge movement to immunofluorescence assays (3, 13, 21, 27, 29). Studies have attributed this effect to the Cav{beta} subunit binding an ER retention signal localized near the AID in the I-II loop of the Ca2+ channel {alpha}1-subunit (4). In particular, the BID of the Cav{beta} subunit present in domain 4 was initially found to be essential for the effect on membrane trafficking of the channel complex (4). However, more recent crystallographic studies (11, 45) suggest that the effects of mutations in the BID are related to alterations in proper protein folding, because the ABP is the actual binding site that recognizes the AID where the ER retention signal is localized. The present immunocytochemistry, surface-membrane biotinylation, and single-channel electrophysiology results demonstrating a lack of membrane trafficking by Cav{beta}1d are consistent with predications that ABP binding to the AID is necessary to relieve ER retention of the {alpha}1-subunit. Two previous studies (25, 26) have characterized functional effects of other short Cav{beta}-splice variants including truncated splice variants of the Cav{beta}3 and Cav{beta}4 genes, but those studies did not directly address membrane trafficking. It is likely that all short Cav{beta} subunits will share the inability to promote membrane trafficking given the lack of the ABP.

A number of different signaling pathways affect the trafficking of Ca2+ channels to the surface membrane by interacting with Cav{beta} subunits. For example, the Ras-related small G protein Gem/Kir can markedly downregulate the surface expression of Cav1.3 L-type Ca2+ channels in endocrine cells (2). Other related proteins in this family including the Rem and Rad GTPases share the ability to remarkably reduce if not eliminate expressed Ca2+ channel activity in a Cav{beta}-subunit-dependent manner (19). In addition, neuronal Ca2+ sensor-1 (NCS-1) can also downregulate the expression of voltage-gated Ca2+ channels in a Cav{beta}-subunit-specific fashion (39). However, the effects of NCS-1 on Ca2+ currents have varied among different experimental systems with some studies showing that NCS facilitates or increases P/Q- and N-type Ca2+ currents, which may be important for activity-dependent synaptic facilitation or synaptic neurotrophic effects (44, 48). Ultimately, understanding how these accessory proteins modulate channel expression and function will require more detailed understanding of how they interact with Cav{beta} subunits including determining what region of the Cav{beta} subunit is required for these effects, how different Cav{beta} subunits vary in response, and in particular, whether the short Cav{beta} subunits such as Cav{beta}1d are capable of modulating this process perhaps by binding to the regulatory proteins without affecting the trafficking of the channels. Therefore, although the short Cav{beta} subunits may not directly affect membrane trafficking of the channel complex, they may act as a sink for modulatory molecules binding to Cav{beta} subunits that could affect trafficking.

Functional effects of short Cav{beta} subunits on Ca2+ channel gating. Although our data suggest that Cav{beta}1d has no detectable effect on membrane trafficking of the {alpha}1C-complex, we provide evidence that the Cav{beta}1d subunit can still modulate the gating of the channels by increasing the mean Po. Comparably, a recent study (26) examining the effect of coexpression of a truncated Cav{beta}3 subunit lacking the ABP with {alpha}1C-subunits also demonstrated an increase in single-channel activity. These results demonstrate that it is possible to separate the effects of the Cav{beta} subunit on membrane trafficking from its ability to modulate channel gating. Even with full-length Cav{beta} subunits, a previous study (21) has shown that the major functional effects of Cav{beta} subunits can be separated from membrane-trafficking effects by mutating a critical residue in the AID in the domain I-II loop of {alpha}1C-subunits. In that heterologous expression study, Cav{beta}-subunit coexpression failed to traffic the AID mutated {alpha}1C-subunit to the surface membrane, but the Cav{beta} subunit was clearly able to modulate single-channel gating (21). In addition, in another study (51) in which Cav{beta}3 fusion proteins were injected into oocytes, the investigators showed that alterations in channel gating occurred much more rapidly than the effects on membrane trafficking. These authors likewise concluded that channel-gating and membrane-trafficking effects could be separately controlled by Cav{beta} subunits.

The modulation of gating of {alpha}1C-channels by the short Cav{beta}1d subunit suggests that sites of interaction between {alpha}1C- and Cav{beta}1d subunits exist besides the well-characterized binding of {alpha}1C-AID in the I-II linker to the Cav{beta} ABP in domain 4. Previous studies (46, 47) have described interactions between the carboxyl terminus of Cav{beta}4 and both the amino and carboxyl termini of the Cav2.1 channel. These interactions, which do not involve the AID and ABP, have generally been found to be of much lower affinity. Additionally, others have argued that the association of the Cav{beta} subunits with {alpha}1C-channels is dynamic and reversible, so multiple low-affinity sites may allow for specific modulation (5, 38). Our data with the short Cav{beta}1d subunit provide evidence for an uncharacterized interaction site that occurs in the amino terminal half rather than the carboxyl terminal portion of the Cav{beta}1 subunit. Furthermore, the interaction site does not involve the first 13 amino acid residues of Cav{beta}1. Lower affinity interactions by the short Cav{beta} subunits may also help to explain the apparent discrepancy between the lack of whole cell electrophysiology effects and the increase in mean Po observed in single-channel measurements observed in our study and that of Hullin et al. (26).

The functional impact of short Cav{beta} subunits such as Cav{beta}1d in cell physiology may also be related to their ability to interact with other Cav{beta} subunits or even other proteins independent of the channel complex. The modular Cav{beta} subunit, like other MAGUKs, can undergo intermolecular interactions that involve SH3 and GK domains (32, 42). Therefore, it is possible that short Cav{beta} subunits interact with full-length Cav{beta} subunits to ultimately affect channel gating and trafficking. In addition, the recently described (25) regulation of gene expression by short splice variants of the Cav{beta}4 gene provides evidence for other important roles of these proteins. Future studies are needed to define the full array of functional interactions of short Cav{beta} subunits in cell physiology.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants R01 HL-61537 (to T. J. Kamp) and PO1 HL-47053 (to T. J. Kamp and J. W. Hell).


   ACKNOWLEDGMENTS
 
The authors acknowledge the assistance of Thankful Sanftleben with manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. J. Kamp, H6/343 Clinical Science Center, Box 3248, 600 Highland Ave., Madison, WI 53792 (E-mail: tjk{at}medicine.wisc.edu)

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.

* R. M. Cohen and J. D. Foell contributed equally to this work. Back


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 GRANTS
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J. Leroy, M. S. Richards, A. J. Butcher, M. Nieto-Rostro, W. S. Pratt, A. Davies, and A. C. Dolphin
Interaction via a Key Tryptophan in the I-II Linker of N-Type Calcium Channels Is Required for {beta}1 But Not for Palmitoylated {beta}2, Implicating an Additional Binding Site in the Regulation of Channel Voltage-Dependent Properties
J. Neurosci., July 27, 2005; 25(30): 6984 - 6996.
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