Multiple Modulation Pathways of Calcium Channel Activity by a beta  Subunit
DIRECT EVIDENCE OF beta  SUBUNIT PARTICIPATION IN MEMBRANE TRAFFICKING OF THE alpha 1C SUBUNIT*

Hiroshi YamaguchiDagger §, Mitsuyoshi HaraDagger , Mark StrobeckDagger , Kenji Fukasawa, Arnold SchwartzDagger , and Gyula VaradiDagger parallel

From the Dagger  Institute of Molecular Pharmacology and Biophysics and the  Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0828

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In order to study the precise mechanisms of alpha 1 subunit modulation by an auxiliary beta  subunit of voltage-dependent calcium channels, a recombinant beta 3 subunit fusion protein was produced and introduced into oocytes that express the human alpha 1C subunit. Injection of the beta 3 subunit protein rapidly modulated the current kinetics and voltage dependence of activation, whereas massive augmentation of peak current amplitude occurred over a longer time scale. Consistent with the latter, a severalfold increase in the amount of the alpha 1C subunit in the plasma membrane was detected by quantitative confocal laser-scanning microscopy after beta 3 subunit injection. Pretreatment of oocytes with bafilomycin A1, a vacuolar type H+-ATPase inhibitor, abolished the increase of the alpha 1C subunit in the plasma membrane, attenuated current increase, but did not affect the modulation of current kinetics and voltage dependence by the beta 3 subunit. These results provide clear evidence that the beta  subunit modifies the calcium channel complex in a binary fashion; one is an allosteric modulation of the alpha 1 subunit function and the other is a chaperoning of the alpha 1 subunit to the plasma membrane.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Voltage-gated calcium channels are heteromultimeric protein complexes that consist of at least three (alpha 1, alpha 2/delta , and beta ) subunits and play a central role in diverse biological functions such as excitation-contraction coupling, excitation-secretion coupling, neurotransmitter release, and regulation of gene expression. The alpha 1 subunit accommodates the channel pore, voltage sensor, and binding sites for various channel-modifying compounds. It has also been shown that the basic characteristics of the different channel types (L-, N-, P-, Q-, and R-type) are carried by the corresponding alpha 1 subunits (1). Auxiliary subunits (alpha 2/delta , beta , and gamma ) modulate calcium channel characteristics, such as current amplitude, voltage dependence, and kinetics of activation and inactivation as well as sensitivity to calcium channel antagonists (2-4).

The modulatory effects of the beta  subunit on the Ca2+ channel complex have been extensively studied using coexpression of cDNAs in heterologous systems. It is well established that coexpression of the alpha 1 subunit with a beta  subunit results in an increase of peak current density (5), acceleration of activation and inactivation kinetics, a leftward shift of the current-voltage relationship, and increased dihydropyridine (DHP)1 binding activity (6-12). However, there have been inconsistencies in the reported mechanism(s) by which these effects occur. Varadi et al. (6) reported a 10-fold increase in the number of DHP-binding sites by coexpression of the alpha 1 subunit with the beta  subunit, suggesting an increase in available channels within the plasma membrane. In contrast, an increase in current amplitude without affecting charge movement by beta  subunit coexpression was shown by Neely et al. (8). These same authors later reported two modes of activation of the alpha 1C subunit (13). Coexpression of a beta  subunit potentiated current by an increase of the fast-activating component, an acceleration of the slow component, and a larger proportion of long openings. An increase in DHP binding and current density without a change in the amount of alpha 1 subunit protein in the plasma membrane was reported by Nishimura et al. (10). These reports suggest that the beta  subunit modulates channel properties by "assisting" the alpha 1 subunit in establishing a proper conformation suitable for a functional Ca2+ channel, rather than affecting expression, trafficking, or stability of the alpha 1 subunit (reviewed by Catterall (14)).

By using immunocytochemical methods, Chien et al. (15) showed that the beta 2a subunit acts as a chaperone-like molecule to facilitate membrane targeting of the alpha 1C subunit, without affecting the total amount of expressed alpha 1C subunit in human embryonic kidney cells. Coexpression of the alpha 1C subunit with beta 2a resulted in a marked increase in localization of the alpha 1C subunit to the plasma membrane. These observations were confirmed by Brice et al. (16) who coexpressed the alpha 1A with alpha 2/delta and several different beta  subunits. By using Xenopus oocytes as an expression system, Shistik et al. (17) reported opposite results in that no change in the amount of alpha 1 subunit protein was found in the plasma membrane when coexpressed with a beta  subunit. Interestingly, an increase in charge movement has been reported when alpha 1C and beta  subunits were coexpressed in a mammalian cell system (18, 19). These discrepancies have been attributed to differences in expression systems employed (20). Taken together, the mechanisms by which the beta  subunit modulates calcium channel activity remain unclear. The limitation of previous studies are that they were all done using coexpression of the alpha 1 and beta  subunits, which makes it difficult to answer the question as to which level of the biosynthesis of the channel complex is the beta  subunit working, i.e. translation, trafficking, and/or direct binding to the membrane-incorporated alpha 1 subunit.

We address these questions by injecting a recombinant beta  subunit protein into Xenopus oocytes expressing the alpha 1C subunit and observing the time course of modulation. We found that changes in voltage dependence of activation and kinetics occurred at an earlier stage in the time course, compared with increases in current amplitude which required a substantially longer time to reach plateau. This suggests that the effects are functionally uncoupled and occur by distinct mechanisms. By using confocal laser-scanning microscopy (CLSM), we found that the amount of the alpha 1 subunit in the plasma membrane was substantially increased by the beta  subunit. Furthermore, pretreatment of oocytes with bafilomycin A1, a vacuolar type H+-ATPase (V-ATPase) inhibitor (21), inhibited the beta  subunit effect on current amplitude, abolished the increase in channel amount in the plasma membrane, but did not influence the beta  effects on voltage dependence and current kinetics.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Bacterial Production and Purification of beta 3 Subunit Fusion Protein-- The human calcium channel beta 3 cDNA clone (22) was subcloned into pBluescript SK(+) between the HindIII and BamHI sites. This plasmid was cleaved with HindIII, and the protruding end was filled in with T4 DNA polymerase and then cut with BamHI to liberate the beta 3-coding fragment. The pET15(b) vector (Novagen) was cleaved with NdeI, filled in with T4 DNA polymerase, and then cut with BamHI. Finally, the beta 3 fragment was ligated into the blunt end/BamHI sites. This strategy has generated an in-frame cloning of the beta 3 protein with the His6-tag sequence and resulted in a fusion product that had the following peptide fused to the N-terminal sequence of the human beta 3 protein, MGSSHHHHHHSSGLVPRGSHKLDP. The sequence of the construct was verified by sequencing through the junction regions.

Escherichia coli BL21(lambda DE3) (Novagen) cells were transformed with the above construct and used for mass production of the His6-tagged beta 3 fusion protein. The cells were cultured in 500 ml of LB medium in the presence of 50 µg/ml ampicillin and grown at 37 °C until an A600 of 0.8 was reached. The production of fusion protein was induced by 0.4 mM isopropyl beta -D-thiogalactopyranoside (IPTG) for various times at temperatures between 23 and 37 °C (we found induction being optimal at 23 °C for 8 h). The cells were pelleted by low speed centrifugation, washed with buffer A (50 mM Tris-HCl, 2 mM EDTA, pH 8.0), resuspended in 10 ml of binding buffer (20 mM Tris-HCl, 1 mM imidazole, 500 mM NaCl, pH 7.9), and disrupted by sonication. The lysate was further fractionated by differential centrifugation. The resulting pellet (1,000 × g, P12) contained nondisrupted cells and inclusion bodies. The supernatant was further centrifuged at 10,000 × g, resulting in a pellet (P3) and a clear supernatant fraction (S), which was purified on a His-bound column (Ni2+-agarose, Novagen). The column was washed with 60 mM imidazole, pH 7.9, and finally the specifically bound protein was eluted with 1 M imidazole and 500 mM NaCl, pH 7.9. We observed that the eluted protein product aggregated when stored frozen in the elution buffer. To prevent aggregation we dialyzed the eluate gradually against 100 mM EDTA·Tris, pH 7.6, and stored at -80 °C in small aliquots. The protein fractions and purification products were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. Protein concentrations were determined with the Pierce protein assay kit.

In Vitro Translation of the hbeta 3 Subunit and Binding of GST Fusion Proteins-- The I-II, II-III, and III-IV intracellular loops were extracted from the human heart calcium channel cDNA (23) via PCR. By using PCR we performed site-directed mutagenesis, placing unique BamHI and XhoI restriction sites at both the 5' and 3' ends of these fragments. The PCR products containing the I-II loop (nucleotides 1089-1499), the II-III loop (nucleotides 2133-2633), and the III-IV loop (nucleotides 3402-3574) were subcloned into the pGEX-4T-1 vector (Amersham Pharmacia Biotech), which is under the control of the tac promoter and contains the glutathione S-transferase (GST) tag. The resulting subclones, pGEXI-II, pGEXII-II, and pGEXIII-IV, were sequenced, and analysis confirmed the presence of the intracellular loops and the absence of additional mutations. The pGEX-4T-1, pGEXI-II, pGEXII-III, and pGEXIII-IV plasmids were transformed into Novablue (DE3) cells (Novagen). Production and purification of GST fusion proteins were done according to the manufacturer's protocol.

The pET15(b) plasmid carrying the hbeta 3 subunit cDNA was used to synthesize hbeta 3 using the coupled in vitro transcription/translation reticulocyte lysate system (Promega) in the presence of 35S-labeled methionine (Amersham Pharmacia Biotech), and a protease inhibitor mixture (1 µg/ml chymostatin, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml pepstatin A, 1 µg/ml phenylmethylsulfonyl fluoride) was used to minimize proteolysis.

Screening of fusion proteins with the radioactively labeled hbeta 3 subunit was performed three times, as described previously (24, 25). Bacterial colonies containing the plasmids were grown overnight at 37 °C. A 1:10 dilution was made in fresh media and grown at 37 °C for ~2-4 h, until an A600 of 1.0 was reached. The bacterial cultures were then induced with 1 mM IPTG for 4 h at 23 °C. Cultures were sedimented by centrifugation and resuspended in ice-cold 1× PBS, 1% Triton X-100 buffer and disrupted by sonication for ~20 s. After sedimentation of the lysate by centrifugation at 4 °C, the soluble fractions containing the GST fusion proteins were adsorbed to glutathione-bound Sepharose (1 volume lysate, 0.1 volume of 50% (v/v) slurry of glutathione-Sepharose 4B (Amersham Pharmacia Biotech)) and incubated for 5 min at room temperature. The slurry was centrifuged at 4 °C and washed 3 times with 1× PBS, 1% Triton X-100 and resuspended in an equal volume of ice-cold 1× PBS, 1% Triton X-100, and 35S-labeled hbeta 3 subunit was added in vitro. This mixture was incubated for 30 min at room temperature. The slurry was centrifuged and washed 4 times with 1× PBS, 1% Triton X-100 and eluted with 10 mM glutathione, 50 mM Tris-HCl, pH 8.0 (Amersham Pharmacia Biotech). After elution, the slurry was spun for 5 min at 4 °C, and the supernatant was removed and added to an equal volume of 2× SDS sample buffer. One-half of the eluate was run on a 5-14% gradient SDS-PAGE gel (Bio-Rad) and stained with Coomassie Blue for protein detection, and the remainder of the eluate was run on another 5-14% gradient SDS-PAGE gel. This gel was fixed, treated with Amplify (Amersham Pharmacia Biotech) for fluorography for 30 min, dried, and exposed to Hyperfilm MP (Amersham Pharmacia Biotech).

Electrophysiology-- Xenopus laevis oocytes (stage V-VI) were prepared as described previously (26). Capped cRNA was synthesized from XbaI-linearized human heart L-type calcium channel alpha 1 subunit DNA template (23) and injected into the oocytes (50 nl, 0.2 µg/µl). After 4-5 days of incubation at 19 °C in the solution containing (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, 2.5 sodium pyruvate, 0.5 theophylline, pH 7.5, supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin, oocytes were injected with 50 nl of either beta 3 subunit fusion protein solution (90 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 10 mM EDTA, 340 ng/µl protein, pH 7.4, with Tris) or vehicle (same composition except protein). The final concentrations in the oocytes were 300 nM beta 3 subunit fusion protein, 4.5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 0.5 mM EDTA, assuming the volume of oocytes was 1 µl. We found this was a saturating amount of protein since in our preliminary experiments higher concentrations did not result in greater effects. In the experiments indicated, oocytes were preincubated in a solution containing 500 nM bafilomycin A1 for 2-3 h prior to the injection of the protein. After injection, they were further incubated in the presence of bafilomycin A1 until currents were measured. Bafilomycin A1 was dissolved in dimethyl sulfoxide as a 1 mM stock solution. The final concentration of dimethyl sulfoxide was 0.05%, which had no effect on the currents or the beta  effect.

The currents were recorded 1-4 h after injection with the beta  protein or vehicle using the standard two-electrode voltage-clamp technique at room temperature (20-21 °C). One oocyte was used for only one recording. The recording medium contained (in mM): 40 Ba(OH)2, 50 N-methyl-D-glucamine, 2 KOH, 5 HEPES, 0.5 niflumic acid, pH 7.4, with methanesulfonic acid. The voltage recording electrode and the current injection electrode were filled with M KCl and had resistances of 0.5-1 MOmega . Currents were recorded using an Axoclamp-2A (Axon Instruments) amplifier. Pulses were applied from a holding potential of -80 mV every 15 s. Whole cell leakage and capacitative currents were digitally subtracted using the P/4 protocol. Data were filtered at 1 kHz, sampled at 1-10 kHz, and stored on a hard disk. Batches of oocytes that showed significant (>20 nA) endogenous Ca2+ channel current were excluded from the analysis. The unpaired t test and analysis of variance were performed for the statistical analyses.

Immunofluorescence Studies-- DNA sequence complementary to the YPYDVPDYA epitope sequence of influenza virus hemagglutinin (HA) (27), which is recognized by the 12CA5 mouse monoclonal antibody, was introduced to the N terminus immediately after the initiator methionine of human heart L-type calcium channel alpha 1C subunit DNA by PCR-based mutagenesis. The cRNA of the epitope-tagged alpha 1 subunit was transcribed and injected into X. laevis oocytes to express tagged channels. The functional characteristics of HA epitope-tagged alpha 1 were tested in a separate set of experiments, and we found that the expressed currents and modulation of them by the beta 3 subunit were indistinguishable from those of wild type (data not shown). After 4-5 days of incubation, oocytes were injected with the beta 3 subunit fusion protein as described above. Four hours after injection, oocytes were fixed with 3.7% formaldehyde, 0.25% glutaraldehyde, and permeabilized with 0.2% Triton X-100 at room temperature. Oocytes were then post-fixed in 100% methanol at -20 °C overnight, incubated with 2% bovine serum albumin for 1 h at room temperature, followed by incubation with 10 µg/ml fluorescein-conjugated anti-HA monoclonal antibody (Boehringer Mannheim) at 4 °C overnight. Oocytes were then washed extensively in PBS and examined by CLSM.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Production and Purification of a Recombinant Human beta 3 Subunit Fusion Protein-- The human beta 3 subunit fusion protein was purified from the soluble fraction of a bacterial culture induced with IPTG at 23 °C for 8 h. The soluble fraction was then applied to a Ni2+-agarose column. Approximately 88.5% of total protein was in the flow-through. A smaller amount of total protein (~8%) weakly bound to the resin and was washed out by 60 mM imidazole. Approximately 3.5% of the total protein bound firmly to the Ni2+-agarose and was eluted with 1 M imidazole and 0.5 M NaCl (Fig. 1A).


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Fig. 1.   Purification of human beta 3 subunit fusion protein. A, purification on Ni2+-agarose column and elution profile of proteins. B, purity of the human beta 3-chromatographic fractions. Lanes 1 and 8, molecular weight markers; lane 2, P12 fraction; lane 3, P3; lane 4, S fraction; lane 5, flow-through; lane 6, waste with 60 mM imidazole; lane 7, pure beta 3 eluted with 1 M imidazole + 0.5 M NaCl. 5 µl of fractions were separated on 10-20% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue.

Analysis of the P12 fraction showed large quantities of the 57-kDa beta 3 protein, which was insoluble and not suitable for purification. The P3 and S fractions contained sizable amounts of the beta 3 protein. Ni2+-agarose affinity purification of the S fraction resulted in highly pure beta 3 protein (estimated purity is higher than 99.5%) with the predicted molecular weight (Fig. 1B, lane 7).

In Vitro Binding of the beta 3 Subunit to the I-II Intracellular Loop-- To determine whether the recombinant His6-tagged beta 3 subunit was able to interact with the calcium channel intracellular I-II loop, as identified by Pragnell et al. (28), we created GST fusion proteins of the I-II, II-III, and III-IV loops of the alpha 1C subunit and screened with an in vitro transcribed and translated, 35S-labeled His6-tagged beta 3 subunit. The data from these experiments clearly show that the His6-tagged beta 3 subunit is able to interact with the I-II loop in a highly specific manner (Fig. 2A). In addition, these results indicate that the beta 3 subunit did not interact with the control GST, II-III, and the III-IV loops. A sample of the eluate was run on an 4-15% SDS-PAGE gel and stained with Coomassie Blue to determine whether the purified fusion protein could also be eluted from the glutathione-Sepharose. All four GST fusion proteins were purified and present in the binding assay eliminating the possibility of nonspecific interaction with other protein components (Fig. 2B). These experiments confirm that the His6-tagged beta 3 subunit maintains its ability to interact with the I-II loop of the alpha 1C subunit in vitro.


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Fig. 2.   Determining human beta 3 subunit interaction with the calcium channel intracellular loops. Elution of GST-bound fusion proteins from glutathione-Sepharose resulted in a sample that was split in half and run simultaneously on separate gels. The first gel was used for autoradiography, and the other was stained with Coomassie Brilliant Blue. A, shown is the autoradiogram of a 4-15% SDS-PAGE gel run with samples of eluate from GST fusion proteins screened with an in vitro transcribed and translated 35S-labeled hbeta 3 subunit. Lane 1, aliquot of 35S-labeled hbeta 3; lane 2, GST control; lane 3, I-II loop; lane 4, II-III loop; lane 5, III-IV loop. B, displays a 4-15% SDS-PAGE gel stained with Coomassie Brilliant Blue. Lane 2, GST-control; lane 3, I-II loop; lane 4, II-III loop; lane 5, III-IV loop.

Electrophysiological Properties of Expressed Human alpha 1C Subunit in Xenopus Oocytes and Time-dependent Effects of Injected Human beta 3 Subunit Fusion Protein-- Oocytes were injected with human alpha 1C subunit cRNA and incubated for 4-5 days. The control (alpha 1C subunit alone) Ca2+ channels expressed in oocytes showed peak barium currents of 149 ± 14 nA (n = 14) when depolarized from a holding potential of -80 mV to test potentials between -30 and +60 mV. The control current exhibited a slow activation and very little inactivation (Fig. 3A), in agreement with previous studies (2, 9). The threshold potential for current activation was found between -20 and -10 mV, and the current peaked at +40 mV. After injection of the beta 3 subunit fusion protein, an increase in current amplitude, a change in the voltage dependence of activation, and changes in activation and inactivation kinetics occurred in a time-dependent manner (Fig. 3A). After injection of the beta 3 subunit fusion protein the activation threshold shifted to hyperpolarizing potentials between -30 and -20 mV, and the current peaked between +20 and +30 mV. The current-voltage (I-V) relationships corresponding to the current traces depicted in Fig. 3A are superimposed in Fig. 3B. Most notably, the shift of the I-V curve occurs within 2 h, whereas the increase in current amplitude requires a longer time scale.


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Fig. 3.   Effects of beta 3 subunit fusion protein on the Ba2+ current through the expressed alpha 1C subunit. A, the representative whole cell current traces for the control (alpha 1C subunit alone) and after the injection of beta 3 subunit fusion protein or vehicle are shown. All traces in this figure were recorded from the same batch of oocytes. Currents were elicited by 1000-ms command pulses from a holding potential of -80 mV to indicated potentials. B, current-voltage relationship corresponding to the traces shown in A. The peak Ba2+ current amplitude was plotted against each test potential. The curve for vehicle-injected oocyte is not shown in order to keep clarity.

Effects of beta 3 Subunit Fusion Protein on Peak Current Amplitude and the Influence of Bafilomycin A1 Treatment-- Fig. 4 depicts the average time course of peak Ba2+ current amplitude enhancement after injection of the beta 3 subunit fusion protein. We observed an increase in peak current amplitude, 2.3-fold at 1 h and 2.9-fold at 2 h after injection of the beta 3 subunit. However, more than half of the increase occurred after 2 h, reaching a plateau at 3 h (6.5- and 6.6-fold for 3 and 4 h after injection, respectively). The effect of the beta  subunit on the current amplitude is clearly slower than the effect on the voltage dependence of activation (cf. Fig. 5). The time of the half-maximal effect for the increase in current amplitude was between 2 and 3 h. Injection of the vehicle had no significant effect on peak current amplitude, although after 4 h the current amplitude appeared to be smaller in some cases. We attribute this to a nonspecific mechanistic disruption by the injection itself.


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Fig. 4.   Time courses of the changes in peak Ba2+ current amplitude after injection of beta 3 subunit fusion protein or vehicle. Currents were elicited by depolarizing pulses from a holding potential of -80 mV. Data points represent averaged values from 5 to 14 experiments. Error bars show S.E. (if not visible, values are within the symbols). Baf, bafilomycin A1-treated group. *, p < 0.05 versus vehicle injected group.


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Fig. 5.   Time-dependent effect of beta 3 subunit fusion protein on the voltage dependence of activation. A, steady-state activation curves for control (alpha 1C subunit alone) and after the injection of beta 3 subunit fusion protein. Fractions of activated channels were calculated by fitting individual experiments to the following equation: I = Gmax(Vm - Vrev)/(1 + exp(V0.5 - Vm)/k), where Gmax is the maximum conductance; Vm is the test potential; Vrev is the current reversal potential; V0.5 is the half-activation potential; and k is the slope factor. The curves show Boltzmann distributions that best fit the averaged data. Average and S.E. from 5 to 8 experiments for each group are shown. B, time courses of the beta  subunit effect on the half-activation potential. Average values from 4 to 14 experiments are shown. Error bars show S.E. Baf, bafilomycin A1-treated group. Half-activation potential was calculated by the above equation.

It has been established that the V-ATPase is responsible for the maintenance of the luminal acidic environment within cell organelles including the Golgi complex, lysosomes, and endosomes (29). Inhibition of the V-ATPase by bafilomycin A1 impairs the intracellular transportation of glycoproteins via alkalinization of the organelles (30). Thus, if the beta  subunit assists the translocation of the alpha 1 subunit to the plasma membrane, inhibition of this pathway may be imposed by consequences on one or all of the auxiliary beta  subunit modulatory function(s). Therefore, we pretreated oocytes with bafilomycin A1 to determine whether the beta  subunit participates in the intracellular translocation of the alpha 1C subunit. Pretreatment of oocytes expressing the alpha 1C subunit with bafilomycin A1 for 2-3 h showed a slight decrease in the control (alpha 1C subunit alone) current (114 ± 14 nA, n = 7) compared with the untreated control. However, these data were not statistically different (p = 0.13 versus untreated control). The effect of the beta 3 subunit protein on peak current amplitude was effectively blocked by preincubation with bafilomycin A1 prior to subunit injection. Only 1.5-, 1.8-, 1.8-, and 1.7-fold current increase was observed at 1-4 h after injection of the beta 3 subunit protein, respectively.

Effects of beta 3 Subunit Protein on the Voltage Dependence of Activation-- Steady-state activation curves were derived from I-V relationships, and the beta 3 subunit effect on the voltage dependence of activation was further analyzed (Fig. 5A). Control currents showed a shallow activation curve with a half-activation potential of 29.9 ± 1.5 mV (n = 14). Injection of the beta 3 subunit protein caused a leftward shift of the curve and an increase in the slope. Most dramatic changes occurred within 1 h, and no significant changes were observed between 2, 3, and 4 h after injection. As shown in Fig. 5B, ~70% of the negative shift in the half-activation potential occurred within 1 h after injection, reaching a plateau at 2 h. The time of the half-maximal effect was less than 1 h. The vehicle had no effect on the voltage dependence of activation. Bafilomycin A1 treatment did not influence the ability of the beta 3 subunit to shift the half-activation potential in the negative direction (Fig. 5B).

Effects of the beta 3 Subunit Protein on Current Kinetics-- We measured the time to half-peak current as a parameter of the macroscopic activation kinetics. As shown in Fig. 3A, the control (alpha 1 subunit alone) current showed slow activation. When depolarized to +30 mV from a holding potential of -80 mV, the time to half-peak was 19.2 ± 1.0 ms (n = 9) (Fig. 6A). The injection of oocytes with the beta  subunit protein rapidly facilitated activation kinetics, reaching a plateau within 2 h, with the time of the half-maximal effect occurring within 1 h. The vehicle did not change activation kinetics. Pretreatment of oocytes with bafilomycin A1 did not change activation kinetics of control (alpha 1 subunit alone) currents and did not modify acceleration of activation kinetics by the beta  subunit protein (Fig. 6A).


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Fig. 6.   Time-dependent effect of beta 3 subunit fusion protein on the current kinetics. A, time courses of the beta  subunit effect on the activation kinetics. Time to half-peak current was measured in current traces that were induced in response to the depolarizing pulses from -80 to 30 mV. Averaged value from 4 to 9 experiments are shown. Error bars show S.E. Baf, bafilomycin A1-treated group. *, p < 0.05 versus vehicle injected group. B, time courses of the beta  subunit effect on the inactivation kinetics. Inactivated fraction at 1 s after the beginning of the test pulse (-80 to 30 mV) was measured. Data points are mean values of 4-16 experiments. Error bars show S.E. Baf, bafilomycin A1-treated group. *, p < 0.05 versus vehicle injected group.

We also investigated the kinetics of inactivation for macroscopic Ba2+ currents. The rate of inactivation was quantitated as the inactivated fraction of Ba2+ current 1 s after the application of a depolarizing test pulse. In control oocytes, the current elicited by the alpha 1 subunit alone showed very little or no inactivation (see Fig. 3A). After injection with the beta  subunit protein the current exhibited a time-dependent faster inactivation (Fig. 6B), similar to the time-dependent changes of voltage dependence and activation kinetics. Again, changes occurred maximally within 2 h, with the time of the half-maximal effect being less than 1 h. The vehicle did not significantly affect the inactivation of Ba2+ current. In addition, pretreatment of oocytes with bafilomycin A1 did not influence the control current nor the effect of the beta  subunit protein on inactivation kinetics (Fig. 6B), suggesting this mechanism of modulation is independent of protein translocation.

beta Subunit Protein Increases the Amount of alpha 1 Subunit in the Plasma Membrane-- We tested whether the injection of the beta 3 subunit protein into oocytes promoted alpha 1C protein delivery to the plasma membrane. The HA epitope-tagged alpha 1C subunit was expressed in Xenopus oocytes that were then injected with the beta 3 subunit protein. We found no difference in either the characteristics of Ba2+ current or effects of the beta 3 subunit protein between the wild-type channels and epitope-tagged channels. The epitope-tagged alpha 1C subunits were detected in the plasma membrane immunocytochemically using CLSM and quantitated by measuring the pixel intensity. When the epitope-tagged alpha 1C was expressed alone in Xenopus oocytes, we observed very little fluorescence in the plasma membrane. In the presence of the beta 3 subunit protein, the amount of the alpha 1C subunit in the plasma membrane increased severalfold (Fig. 7). Furthermore, pretreatment of oocytes with bafilomycin A1 completely abolished this increase, strongly suggesting that this process is dependent on protein translocation. We obtained similar results in 10-15 oocytes for each group over two different batches of oocytes.


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Fig. 7.   Immunofluorescence detection of the alpha 1C subunits in oocyte plasma membrane. HA-tagged alpha 1C subunits were stained with fluorescein-conjugated anti-HA antibody and analyzed by CLSM. Left panels: Control, plain oocyte; alpha 1C, expressing alpha 1C subunit alone; alpha 1C + beta 3 protein, expressing alpha 1C subunit and injected with beta 3 subunit protein; and Baf, alpha 1C + beta 3 protein, expressing alpha 1C subunit, pretreated with bafilomycin A1, and injected with beta 3 subunit protein. Pixel intensity along the straight line (left panels) are plotted in right panels where the abscissa corresponds to the length of the line. Oocytes shown in this figure were derived from the same series of experiment.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Modulation of calcium channel activity by auxiliary subunits has been extensively studied using many different recombinant expression systems. These studies have clearly demonstrated that coexpression of the beta  subunit alters calcium channel characteristics by increasing current density, shifting the voltage dependence of activation and accelerating channel kinetics. Taken together, these modulatory alterations are believed to impart the inherent current characteristics observed in native preparations. An issue that remains unresolved is whether the beta  subunit increases the amount of the alpha 1 subunit in the plasma membrane or modulates calcium channel functions solely through an allosteric pathway. Experimental observations to date present conflicting results. When the alpha 1 subunit was coexpressed with the beta  subunit in Xenopus oocytes, no change in channel expression was observed, when measured as a function of gating charge movement (8). Moreover, in the same experimental system, the amount of 35S-labeled alpha 1 subunit in the plasma membrane did not change (17). Furthermore, Nishimura et al. (10) have also shown no change in the alpha 1 subunit content of membrane fractions by immunoblotting analysis of cells transfected with the beta  subunit. In contrast, several studies using mammalian cells have shown an increase in charge movement when transfected with beta  (18, 19) and the involvement of the beta  subunit in translocation of the alpha 1 subunit to the membrane (15, 16). However, the biochemical mechanism(s) responsible for this modulation have remained unclear due to insufficient experimental methods to monitor time-dependent biological events occurring intracellularly. In an effort to overcome this limitation, and to resolve apparent conflicting data, we expressed the L-type calcium channel alpha 1 subunit in oocytes, and after injecting a highly purified recombinant beta 3 subunit protein, we examined the time-dependent changes to peak current, voltage dependence, and kinetics. Our results demonstrate for the first time a time-dependent uncoupling of beta 3 subunit modulation of voltage dependence and kinetics from enhancement of peak current density. Moreover, these results suggest that beta  subunit modulation occurs via an allosteric mechanism and through facilitation of protein translocation.

beta 3 Subunit Binding to the I-II Intracellular Loop-- In order to determine whether the His6-tagged beta 3 subunit interacts with the alpha 1 interaction domain (AID) of the intracellular I-II loop as described by Pragnell et al. (28), we screened GST fusion proteins containing each of the three intracellular loops I-II, II-III, and III-IV with an 35S-labeled His6-tagged beta 3 subunit. We found that the beta 3 subunit was able to interact with the I-II loop, suggesting that the presence of six histidine residues at N terminus does not interfere with the binding of this subunit to its intracellular binding site. Since Walker and co-workers (31) have shown that the modulatory functions of the beta  subunit are largely dependent on this interaction, and we have shown that the His6-tagged beta 3 subunit can interact with the I-II loop, the beta 3 subunit should modulate calcium channel function when injected into oocytes as a purified fusion protein. It was also clear from the results of these experiments that the beta 3 subunit did not interact with either the intracellular II-III or the III-IV loops. Although our in vitro experiment showed highly specific interaction between the beta 3 subunit and the alpha 1C I-II loop, we cannot exclude possible weak or transient interactions with other intracellular regions.

Injection of Oocytes Expressing the Ca2+ Channel alpha 1 Subunit with a Recombinant beta  Subunit Fusion Protein Induces Effects Comparable to alpha 1-beta Coexpression-- Effects of beta  subunit coexpression on the alpha 1C subunit have been studied using Xenopus oocytes (7, 8, 17, 32, 33) as well as in mammalian cells (6, 9, 10, 15, 18). It is now generally accepted that coexpression of the beta  subunit results in a 2-fold to more than 100-fold increase in peak current amplitude, a hyperpolarizing shift of the voltage dependence of activation, and acceleration of kinetics of the current (to a different extent, depending on the different types of beta  subunits). Since our results showed comparable effects of the beta  subunit on these parameters (e.g. ~6.5-fold increase in current amplitude, ~16 mV hyperpolarizing shift of half-activation potential, and acceleration of activation and inactivation kinetics) within 3 h of beta  subunit protein injection, it is unlikely that the beta  subunit is enhancing protein synthesis of alpha 1 subunits in the endoplasmic reticulum. Employing our experimental conditions, it takes at least 3 days from the time of co-injection of the alpha 1C and beta  subunit cRNAs and at least 4 days for the alpha 1C subunit cRNA alone to get measurable current through these expressed channels. Therefore, even if we assume that the presence of the beta  subunit somehow facilitates protein synthesis of the alpha 1 subunit, its contribution to the observed effects should be minimal, since our recording time scale is shorter. Thus, we believe that the effects of the beta  subunit are exerted mainly on mechanism(s) downstream of protein synthesis.

The Time Course of the beta  Subunit Effects Can Be Categorized in Two Distinct Patterns-- In our present study, we analyzed the time course of four parameters after injection of the beta  subunit protein as follows: 1) peak Ba2+ current amplitude; 2) voltage dependence of activation; 3) activation kinetics; and 4) inactivation kinetics. Among these, only the peak current amplitude increased within a slow time framework; it took 3 h to reach plateau and the time of the half-maximal effect was between 2 and 3 h. The other three parameters behaved very similarly to each other and changed with a faster time course, reaching a steady level in 2 h, and the time of the half-maximal effect was less than 1 h. According to the in vitro binding study by De Waard et al. (24), AID and the beta 1b subunit bound at a rate constant of 0.1 min-1·µM-1, and when using 500 nM AID, the rate constant corresponded to a half-time of about 20 min. Since our "faster" time course falls within the same time range as their results, we believe that the faster time course indicates direct binding of the injected beta 3 subunit protein to the alpha 1C subunits that already exist in the plasma membrane. We do not know the exact time it takes for the injected beta 3 subunit protein to diffuse, reach the plasma membrane, and build to a saturated concentration. However, since an excess amount of beta 3 subunit protein was injected (final concentration in the oocyte cytoplasm was 300 nM, which is about 5.4 times higher than the reported Kd of AID and beta 3 subunit by De Waard et al. (24), 55.1 nM), we assume it takes less than 1 h. Taken together, it seems that the binding of the injected beta 3 subunit protein to the alpha 1C subunits in the plasma membrane reaches equilibrium within 2 h. Bafilomycin A1 treatment did not influence the beta  effects that are categorized with a faster time course but did abolish the increase of alpha 1 subunit by the beta  subunit. This finding also supports the concept that allosteric modulation of alpha 1 subunits in the plasma membrane by the beta  subunit is responsible for the faster effects, whereas the "slower" component implies the existence of a distinct mechanism for modulation. In order to clarify this, we addressed the question whether the beta  subunit modulation of current amplitude occurs during protein translocation.

Influence of the Pretreatment with Bafilomycin A1-- Bafilomycin A1, a V-ATPase inhibitor, inhibits intracellular glycoprotein transport by impairing the acidification of organelles (30). In the presence of this compound the beta  subunit failed to increase the amount of alpha 1 subunit in the plasma membrane. Bafilomycin A1 also significantly blocked the effect of the beta  subunit on current amplitude. The results strongly suggest that the effect of the beta  subunit protein on current amplitude is largely dependent on intracellular translocation of nascent alpha 1 subunits. The beta  subunit elicited a ~1.8-fold increase of current when oocytes were treated with bafilomycin A1, despite a complete loss of increase of the alpha 1 subunit in the plasma membrane. An attractive explanation for the current increase is that the injected beta  subunit allosterically modulates the population of alpha 1 subunits that are already inserted in the membrane. This is in agreement with single channel analyses done by Wakamori et al. (33), in which the coexpression of the alpha 1 subunit with the beta  subunit resulted in ~2-fold increase in the channel open probability.

In the present study, pretreatment with bafilomycin A1 slightly decreased the control (alpha 1 subunit alone) current amplitude, without affecting other characteristics of the current. Assuming that there is turnover of channels in the plasma membrane (15), some breakdown of functional channels will occur and will be replaced by the translocation of new channels from the cytosolic region. Therefore, it seems reasonable that inhibition of protein translocation results in a decreased number of functional channels and, consequently, decreased current amplitude. The contribution of other mechanism(s), such as destabilization of membrane-incorporated channels, cannot be excluded.

Possible Role of Xenopus Oocyte Endogenous beta  Subunit-- Recently Tareilus et al. (25) cloned an endogenous beta  subunit from Xenopus oocytes (beta XO) which is highly homologous to the mammalian beta 3 subunit. Injection of Xenopus beta  antisense oligonucleotides significantly reduced the current through the expressed alpha 1C and alpha 1E subunit of the Ca2+ channel. Based on this, the authors proposed that the "alpha 1 alone" channels are in fact forming an alpha 1 subunit-endogenous beta  subunit complex (alpha 1beta XO). However, it is not possible at this time to determine whether the endogenous beta XO was present in high enough concentration to complex with the alpha 1 subunit in the absence of exogenous beta . Thus, it seems likely that our observations incorporate the effects of exogenous beta 3 subunit on the alpha 1beta XO complex. However, the basic characteristics of the control (alpha 1 alone) current in the present study, viz. slow activation, slow inactivation, smaller current amplitude, and a shifted voltage dependence to the depolarized direction, and the modulation of these parameters by the application of exogenous beta  subunit are in good agreement with previous studies using a variety of mammalian cells (6, 9, 10, 15, 19) in which no endogenous beta  subunits have been reported.

The strategy and molecular reagents used throughout these studies are thought to be required for efficient interaction among all calcium channel alpha 1 and beta  subunits (25, 28, 34, 35), viz. the I-II intracellular connecting loop and C-terminal tail of alpha 1 and a full-length beta  subunit. Therefore, it is reasonable to conclude that the mechanisms described in the present study should apply for all calcium channel alpha 1 and beta  subunit interactions.

In summary, we have provided compelling evidence that the beta  subunit modulates the function of the alpha 1 subunit of the voltage-dependent Ca2+ channel in two distinct modes, i.e. allosteric modulation and chaperoning of channels to the plasma membrane.

    ACKNOWLEDGEMENTS

We thank Gabor Mikala for making the HA epitope-tagged alpha 1 subunit, Ilona Bodi for oocyte preparation, and Udo Klöckner and Howard Motoike for helpful discussions.

    FOOTNOTES

* This work was supported in part by the Naito Foundation (to H. Y.), by a fund from Kansai Medical University (to M. H.), and by National Institutes of Health Grant P01 HL22619-19 (to A. S.).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.

§ Supported by Postdoctoral Fellowship SW-97-35-F from the American Heart Association Ohio-West Virginia Affiliate.

parallel To whom correspondence should be addressed: Institute of Molecular Pharmacology and Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave., P. O.Box 670828, Cincinnati, OH 45267-0828. Tel.: 513-558-2466; Fax: 513-558-1778; E-mail: varadig{at}email.uc.edu.

1 The abbreviations used are: DHP, dihydropyridine; CLSM, confocal laser-scanning microscopy; V-ATPase, vacuolar type H+-ATPase; IPTG, isopropyl beta -D-thiogalactopyranoside; GST, glutathione S-transferase; AID, alpha 1 interaction domain; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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