A beta 4 Isoform-specific Interaction Site in the Carboxyl-terminal Region of the Voltage-dependent Ca2+ Channel alpha 1A Subunit*

Denise WalkerDagger §, Delphine BichetDagger , Kevin P. Campbellpar , and Michel De WaardDagger **

From Dagger  INSERM U464, Institut Fédératif Jean Roche, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France and  Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The voltage-gated calcium channel beta  subunit is a cytoplasmic protein that stimulates activity of the channel-forming subunit, alpha 1, in several ways. Complementary binding sites on alpha 1 and beta  have been identified that are highly conserved among isoforms of the two subunits, but this interaction alone does not account for all of the functional effects of the beta  subunit. We describe here the characterization in vitro of a second interaction, involving the carboxyl-terminal cytoplasmic domain of alpha 1A and showing specificity for the beta 4 (and to a lesser extent beta 2a) isoform. A deletion and chimera approach showed that the carboxyl-terminal region of beta 4, poorly conserved between beta  isoforms, contains the interaction site and plays a role in the regulation of channel inactivation kinetics. This is the first demonstration of a molecular basis for the specificity of functional effects seen for different combinations of these two channel components.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Voltage-dependent calcium channels have been classified into five groups, based on their electrophysiological and pharmacological properties. L-type channels are ubiquitous, present particularly in skeletal and cardiac muscle, where they play an essential role in excitation-contraction coupling. T-type channels are important for cardiac pacemaker activity and the oscillatory activity of several thalamic neurons, while N- and P/Q-type channels are important in the control of neurotransmitter release in the central and peripheral nervous systems, and the role of R-type channels remains unclear. Two of these channels have been purified to homogeneity, the skeletal muscle L-type channel and the brain N-type channel (1, 2). Although these channels differ dramatically in function, their subunit compositions are very similar, the core subunit composition of a high voltage-activated channel consisting of an alpha 1 subunit, the ionic pore of the channel, and two auxiliary subunits, beta  and alpha 2delta , that confer native biophysical and pharmacological properties to the channel. These subunits are encoded by at least six alpha 1, four beta , and one alpha 2delta gene, for which numerous splice variants have been identified (3).

The beta  subunit is a cytoplasmic protein of 52-78 kDa that, when coexpressed with the alpha 1 subunit, results in an increase (of up to 100-fold) in current amplitude, alteration of both the kinetics and voltage dependence of activation and inactivation, and an apparent increase in recognition sites for channel-specific toxins (e.g. see Refs. 4-8). The regulatory effects of beta  vary in importance, depending on the combination of channel subunits studied. Although beta  regulation seems to be highly conserved from beta 1 to beta 4 and on alpha 1S to alpha 1E, some important differences between these various isoforms have nevertheless been noted. The different beta  subunits produce consistently different channel inactivation behaviors, beta 3 producing fast inactivation, beta 2 slow channel inactivation, and beta 1 and beta 4 more intermediate behaviors (9-11). The beta  effect also appears to be alpha 1 isoform-dependent; the beta -induced shift in voltage dependence of inactivation has been reported for non-L-type channels, A, B, and E (4, 12), whereas it is absent for L-type channels, S, C, and D (13). Since the interaction between calcium channel subunits is promiscuous, at least for alpha 1 and beta  subunits (11, 14), the heterogeneity of combinations observed so far in two native channel types (N-type (15) and P/Q-type (16)) must be of functional significance in cell biology.

Recent studies have identified complementary interaction domains on the alpha 1 and beta  subunits (17, 18). AID1 (alpha 1 subunit interaction domain), a highly conserved region in the cytoplasmic loop between transmembrane domains I and II, interacts with a stoichiometry of 1:1 (11) with BID (beta subunit interaction domain), a 30-residue region in the second conserved domain of the beta  subunit (domain IV in Fig. 5A). AID and BID appear to be essential for the subunit interaction and regulation by beta  subunits (17). Point mutations in AID and BID that disrupt this primary interaction also totally inhibit channel regulation by beta , suggesting that it acts as an important anchoring site, due to its very high affinity (11). Several lines of evidence suggest, however, that, despite its importance in channel regulation, the AID-BID attachment site does not account for all of the regulatory potential of the beta  subunit. The deletion approach used to identify the BID site revealed that it may not carry all the current stimulatory function of beta , the change in inactivation kinetics (17), nor the shift in voltage dependence of inactivation.2 It is also interesting that BID represents only 30 residues in a region that shows 78% identity between beta  subunits over 200 residues and that, in addition, toward the amino-terminal of beta  there exists another highly conserved region (65% identity, over more than 100 residues) (17). The high level of sequence conservation is indicative of evolutionary constraint, suggesting that these regions are of functional importance. The remaining three less conserved domains (I, III, and V) undergo splicing and may also be functionally relevant to beta -specific changes in inactivation as suggested by several studies (19, 20).

Viewed together, the inability of BID to account for all of the functional effects of the beta  subunit and the high level of conservation elsewhere in the sequence lead to the hypothesis that there may exist secondary sites of interaction between the alpha 1 and beta  subunits. Such sites would be dependent on the initial, highest affinity, essential interaction between AID and BID, and therefore need not be of high affinity themselves. The current work concerns the identification and characterization of interaction sites for the beta  subunit in the carboxyl-terminal domain of alpha 1A, which is the largest of the cytoplasmic regions and shows considerable degeneracy of sequence homology between alpha 1 subunit types and splice variants. We demonstrate that the carboxyl-terminal sequence of beta 4 specifically interacts with this region of rabbit alpha 1A (BI-2) and is required for a proper regulation of channel inactivation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Preparation of Fusion Proteins-- Regions of the rabbit brain alpha 1A cDNA (BI-2) (21) corresponding to residues 1889-2126, 2090-2424, 2275-2424, 2070-2275, 2070-2196, 2120-2196, and 2197-2275 were amplified by PCR, and, with the aid of BamHI and EcoRI or XbaI restriction sites included in the primers, subcloned into these sites in pGEX2TK or pGEXKG (Pharmacia Biotech Inc.). The resulting recombinant plasmids were expressed in Escherichia coli BL21, and GST fusion proteins were purified as described previously (11). The newly purified fusion proteins are referred to as, for example, GST-1889-2126A. GST alone and a GST fusion protein of the AID region of alpha 1A (11) were prepared in the same way.

In Vitro Translation of beta  Subunits-- beta subunit cDNA clones used were rat beta 1b (L11453), rabbit heart beta 2a (X64297), rabbit heart beta 3 (M88751), and rat brain beta 4 (A45982). Truncated derivatives of beta 4 were constructed by PCR amplification of the corresponding regions of cDNA and subcloning into pCDNA3, using HindIII and BamHI sites (added to the PCR primers), with the addition of a Kozak (22) sequence and initiation codon (ACCATGG) or termination codon (TGA) as necessary. For construction of a chimera between beta 3 (1-360) and beta 4 (402-519), a two-step PCR approach was used with the following primers: beta 3, forward: 5'-ACGTAAGCTTACCATGGATGACGACTCGTAGGTGCCC-3', reverse: 5'-GCTTGTGTGGGTGGCGCGCCAGTAAACCTCTAGGTA-3'; and beta 4, forward: 5'-GAGGTTTACTGGCGCGCCACCCACACAAGCAGTAGC-3', reverse: 5'-CGCGGATCCTCAAAGCCTATGTCGGGAGTCATGGCTGCATCC-3'. The reverse primer for beta 3 and the forward primer for beta 4 contain complementary sequences, allowing annealing of the two PCR products to give the template for the second round of amplification, using the beta 3 forward and beta 4 reverse primers. Restriction sites HindIII and BamHI were included in the external primers, allowing subcloning into pCDNA3.

35S-Labeled beta  subunits were synthesized in vitro using the TNTTM coupled transcription/translation system (Promega). Nonincorporated [35S]methionine was removed by purification on a PD10 column (Pharmacia).

Binding Assays-- Purified GST fusion proteins were coupled to glutathione-agarose beads (Sigma) by incubation for 30 min, before addition of the translation mixture (approximately 500 pM final concentration). Binding assays were carried out in a final volume of 200 µl, in Tris-buffered saline (0.1% Triton X-100, 25 mM Tris, 150 mM NaCl, pH 7.4), at 4 °C, for 6 h, unless otherwise stated. Beads were washed four times in binding buffer, and then analyzed either by SDS-PAGE and autoradiography or by scintillation counting.

A peptide corresponding to the AID site of alpha 1A (RQQIERELNGYMEWISKAE from Genosys) was dissolved in phosphate-buffered saline (154 mM NaCl, 40 mM Na2HPO4, 11.5 mM NaH2PO4, pH 7.4) at 500 µM and added to binding reactions at 100 µM final concentration. Since addition of phosphate-buffered saline to the binding reactions introduced slight changes in binding affinity, an equal volume of phosphate-buffered saline was added to control reactions.

Electrophysiological Recordings-- Stage V and VI Xenopus oocytes were injected with BI-2-specific mRNA (400 ng/µl) in combination with beta 4-specific mRNA (100 ng/µl) or truncated beta 4Delta C mRNA (100 ng/µl). Cells were incubated for 3 days in defined nutrient oocyte medium as described previously (11). Whole cell recordings were performed at room temperature (22-24 °C) using the two-microelectrode voltage clamp configuration of a GeneClamp amplifier (Axon Instruments, Foster City, CA). The extracellular recording solution was of the following composition (in mM): Ba(OH)2 40, NaOH 50, KCl 3, HEPES 5, niflumic acid 1, pH 7.4 with methane sulfonic acid. Electrodes filled with 500 mM cesium acetate, 10 mM EGTA, 3 mM KCl, and 10 mM HEPES, pH 7.2, had resistances comprised between 0.5 and 2 megohms. The bath solution was clamped to a reference potential of 0 mV. Current records were filtered at 1 kHz, leak-subtracted on-line by a P/6 protocol, and sampled at 2-4 kHz. Data were analyzed using pCLAMP version 6.02 (Axon Instruments). All values are mean ± S.D.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Purified GST fusion proteins carrying amino acids 1889-2126 (GST-1889-2126A) and 2090-2424 (GST-2090-2424A) of the alpha 1A subunit carboxyl-terminal region were coupled to glutathione-agarose beads at a concentration of 5 µM and assayed for interaction with a 35S-labeled in vitro translated rat beta 4 subunit (Fig. 1). GST-1889-2126A showed no significant binding, as seen for the control GST protein alone, while GST-2090-2424A showed a significant level of interaction, comparable to that seen for a 500 nM, saturating (11) concentration of a GST fusion protein carrying the AID region of alpha 1A (GST-AIDA).


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Fig. 1.   In vitro binding of 35S-beta 4 to the carboxyl-terminal region of alpha 1A. A, Coomassie Blue-stained SDS-PAGE (9%), showing GST fusion proteins used (5 µg). B, autoradiogram of binding assay. In vitro translated beta 4 (T) was assayed for binding to the fusion proteins indicated (5 µM). Following binding interactions (as described under "Experimental Procedures"), washed beads were resuspended in SDS-PAGE loading buffer.

Analysis of the binding of various concentrations of GST-2090-2424A to 35S-beta 4 (Fig. 2A) demonstrates that binding is saturable; specific binding appears at about 25 nM and saturates at 500 nM. Comparison of the saturation curve of GST-2090-2424A binding to beta 4 to the dose-response curve of GST-AIDA reveals a dissociation constant (Kd) of 93 nM for GST-2090-2424A, which is an approximately 30-fold lower affinity compared with the GST-AIDA-beta 4 interaction. Association kinetics (Fig. 2B) are relatively slow compared with those previously seen for the AID-BID interaction (11), with a half-time of association of approximately 120 min at 5 µM compared with 20 min at 500 nM GST-AIDA.


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Fig. 2.   In vitro characterization of the alpha 1A-beta 4 interaction. A, various concentrations of GST-2090-2424A fusion protein were assayed for binding to 35S-beta 4, and binding was quantified by counting. Specific binding was calculated by subtraction of binding to GST (at the same concentrations) and normalized by expression as a proportion of maximal binding. Error bars represent normalized S.D. The dotted line represents binding of 35S-beta 4 to GST-AIDA under the same conditions, with a Kd of 3 nM, shown for comparison. Data were fitted by a logistic function y = (a - d)/(1 + (x/c)b) + d where a = 1.019 and d = 0.02 (the asymptotic maximum and minimum, respectively); c = 93 nM (the Kd); and b = -1.6 (the slope of the curve). B, kinetics of 5 µM GST-2090-2424A fusion protein binding to 35S-beta 4 subunit at 4 °C. The data were fitted by an hyperbolic function y = (a·t)/(b + t), where t is the time of association before washing, a = 1.34 (the theoretical maximum of binding), and b = 120 min (the time of half-maximum binding). C, fusion proteins (500 nM GST-AIDA, 2.5 µM GST, and 2.5 µM GST-2090-2424A), were assayed for binding to 35S-beta 4 in the presence or absence of 100 µM AIDA peptide and quantified by counting. Results are shown as counts/min; error bars represent S.D.; the asterisk represents a statistically significant effect of the AIDA peptide. D, various concentrations of GST-2090-2424A fusion protein were assayed for binding to 35S-beta 4 in the presence or absence of 100 µM AIDA peptide and quantified by counting. Results were normalized by subtraction of binding to GST under the same conditions and expression as a proportion of maximal binding in the absence of peptide. Error bars represent normalized S.D.

We next tested whether the AID-BID interaction had any effect on the interaction between GST-2090-2424A and beta 4. In the presence of a 21-amino acid synthetic peptide containing the AID sequence of alpha 1A, specific binding of beta 4 to GST-AIDA is diminished by over 90%, demonstrating the effectiveness of the peptide. The same peptide had no significant effect on the binding of GST-2090-2424A to beta 4 (Fig. 2C), however, indicating that the BID region is not implicated in this interaction. The peptide did not modify the maximum binding of GST-2090-2424A, indicating that, at least in vitro, binding of AID to the beta  subunit does not induce conformational changes capable of favoring (or indeed disfavoring) this interaction. This was further investigated by analyzing the effects of AIDA peptide on the binding to beta 4 at various concentrations of GST-2090-2424A (Fig. 2D). The data show that the peptide also had no significant effect on the affinity of beta 4 for GST-2090-2424A.

To identify the region of the carboxyl-terminal domain that interacts with beta 4, we constructed a series of smaller GST fusion proteins encoding smaller fragments of this region (Fig. 3A) and compared their binding to 35S-labeled beta 4 (Fig. 3B). Within the region from residue 2120 to the carboxyl terminus of the molecule, a whole series of subcloned fragments maintained an ability to interact with 35S-beta 4. Further investigations suggested, however, that these interactions occur with a weaker affinity than GST-2090-2424A. For example, we found a Kd of 225 nM for GST-2070-2196A binding to beta 4 (data not shown), i.e. 2-fold lower. These data indicate that a series of "microsites" are responsible for the binding activity of the alpha 1A carboxyl terminus, perhaps together forming a binding pocket, although dependence on overall conformation of the binding domain appears to be limited. This further contrasts with the beta  interaction to AID, which relies on only three crucial AID residues (14).


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Fig. 3.   Localization of the interaction site in alpha 1A. A, above, schematic diagram of alpha 1A. Amino acid positions are shown above, transmembrane domains (each composed of six membrane-spanning segments) are shown as hatched boxes. Below, enlargement of the carboxyl-terminal domain, showing GST fusion proteins constructed, with amino acid positions marked at the extremities. B, capacity of fusion proteins (5 µM) to interact with 35S-beta 4 and binding was quantified both by SDS-PAGE and autoradiography and by beta  counting. T, equivalent volume of in vitro translation of 35S-beta 4 used in binding assays. B, 35S-beta 4 that remained bound following washing of beads. Percentage values indicate specific binding (under the same experimental conditions, quantified by counting) as a percentage of binding to GST-AIDA (500 nM, i.e. at saturation).

All four beta  subunit isoforms show a fairly similar affinity for AID. However, the functional effects of coexpression of these isoforms vary considerably and also depend on the alpha 1 subunit tested. Functional differences among beta  subunits may be a reflection of the differing capacities of beta  isoforms to form secondary interactions with the alpha 1 subunit concerned. We therefore tested whether the interaction observed between beta 4 and GST-2090-2424A also existed for other beta  subunits translated in vitro. Fig. 4 shows a comparison of binding of 35S-beta subunits to three different concentrations of GST-2090-2424A, to GST alone, and to a GST fusion protein expressing AIDA, at a concentration expected to yield maximal binding. beta 4 interacts with GST-2090-2424A with a high affinity, showing maximal binding at 1 µM fusion protein concentration. beta 2a binds with a much lower affinity, showing only limited binding at 10 µM fusion protein concentration, while binding of beta 3 and beta 1b is insignificant even at this concentration of fusion protein.


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Fig. 4.   beta subunit specificity. Fusion proteins (500 nM GST-AIDA, 10 µM GST, and 100 nm GST-2090-2424A, 1 and 10 µM final concentrations, shown from left to right) were assayed for binding to 35S-labeled beta 1b, beta 2a, beta 3, and beta 4. Results are shown as counts/min, with error bars representing S.D.

The differences in interaction affinity observed for different beta  subunits and the fact that the main regions of sequence divergence among beta  subunits are the carboxyl- and amino-terminal regions (Fig. 5A) suggested that one of these regions was responsible for the interaction. We investigated this possibility (Fig. 5B) by deleting either or both regions from the beta 4 cDNA. We assayed the capacity of the resulting in vitro translated proteins to bind to two fusion proteins, GST-2070-2275A and GST-2275-2424A, which represent approximately the two halves of the region of alpha 1A under investigation (Fig. 2B). Deletion of the amino-terminal 48 amino acids of beta 4 had no effect on the binding of GST-2070-2275A and GST-2275-2424A. In contrast, deletion of the carboxyl-terminal 109 amino acids of beta 4 drastically interferes with its capacity to interact with either fusion proteins. Residual weak binding of both fusion proteins seemed to be present. To check whether this residual binding was due to the amino terminus, we tested the binding of these fusion proteins to the double mutant beta 4Delta N,C. The results show that there was no difference between beta 4Delta C and beta 4Delta N,C, confirming the absence of a binding function for the amino terminus. The importance of the carboxyl-terminal region was confirmed by constructing a chimera in which the carboxyl-terminal region of beta 3 was replaced by the corresponding region of beta 4 (Fig. 5B). The inability of beta 3 to interact with either alpha 1A fusion protein was successfully "rescued" by replacement of this region, resulting in a binding capacity approaching that of the full-length beta 4 subunit.


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Fig. 5.   Localization of the interaction site in beta 4. A, map of a beta  subunit, dividing the protein into five domains according to the degree of sequence conservation between isoforms. Percentage identity between beta 3 and beta 4 is shown above, for each domain. Below, map of beta  subunits and derivatives constructed (beta 4, full-length; beta 4Delta C, beta 4 residues 1-409; beta 4Delta N, beta 4 residues 49-519; beta 4 Delta N,C, beta 4 residues 49-409; beta 3, full-length; and beta 3-4chim, chimera between beta 3 residues 1-360 and beta 4 residues 402-519). B, fusion proteins (GST-2070-2275A and GST-2275-2424A, 5 µM) were assayed for binding to in vitro translated beta  subunits and derivatives.

Interestingly, the results obtained with the full-length, truncated, and chimera beta 4 were very similar for the two fusion proteins assayed. This further suggests that the carboxyl terminus of alpha 1A forms a single binding site and not several sites that would interact independently with diverse regions of beta 4.

Since deletion of carboxyl-terminal sequences of the alpha 1C channel induces important modifications in channel gating and opening probability, we determined the functional importance of this alpha 1A-beta 4 interaction by expression in Xenopus oocytes. Comparison of the effects of the full-length beta 4 and beta 4Delta C revealed that the carboxyl terminus of the beta  subunit had little influence on the biophysical properties of the alpha 1A channel with the exception of a role in the control of inactivation kinetics (Fig. 6A). There were no noticeable differences in current amplitude between alpha 1Abeta 4 and alpha 1Abeta 4Delta C-injected cells with average peak amplitudes of 898 ± 686 nA (n = 5) and 626 ± 413 nA (n = 6), respectively. In addition, no differences were detected in activation parameters with half-activation potentials of 1.5 ± 5.5 and 3.2 ± 3.5 mV, and slope values of 4.9 ± 1 and 5.2 ± 0.9 mV for alpha 1Abeta 4 and alpha 1Abeta 4Delta C-injected cells, respectively. We also found no statistical difference in the voltage dependence of inactivation with half-inactivation potentials of -24.6 ± 5.8 mV (n = 5, alpha 1Abeta 4) and -28.1 ± 4.7 mV (n = 6, alpha 1Abeta 4Delta C). Interestingly, there was a small but significant change in the rate of inactivation kinetics produced by the beta 4 carboxyl-terminal deletion. Cells injected with alpha 1Abeta 4 cRNAs inactivated rapidly after depolarization. The decay in current represents the sum of three components at all voltages (Fig. 6B), two of which are exponential, a fast decaying current with an average time constant of 51 ± 5 ms (25.7 ± 2.9% of total current at 20 mV), and a slow inactivating component with a time constant of 241 ± 30 ms (69.7 ± 6% at 20 mV) and a noninactivating current (4.5 ± 4.3%). The truncated beta 4Delta C increased the overall rate of inactivation by two essential modifications: (i) a decrease in the fast time constant to 43 ± 4.7 ms at 20 mV instead of 51 ms (Fig. 6C) and (ii) a change in the ratio between fast and slow inactivating components from 25.7 to 36 ± 5% (fast) and from 69.7 to 60.7 ± 4.2% (slow) (Fig. 6D). Although these effects were small, they were statistically significant and contributed to the overall average increase in channel inactivation as described in Fig. 6E. These results suggest that the carboxyl terminus of beta 4 may actually contribute to a slowing in inactivation kinetics upon association to the carboxyl terminus of alpha 1A.


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Fig. 6.   Electrophysiological analysis of the effects of carboxyl-terminal truncation of beta 4. Modification in channel inactivation induced by carboxyl-terminal truncation of the beta 4 subunit. A, representative current traces of alpha 1Abeta 4 and alpha 1Abeta 4Delta C injected oocytes depolarized to -10, 0, 10, 20, and 30 mV. B, inactivating components of a current trace obtained by depolarizing an alpha 1Abeta 4Delta C cell to 20 mV. Data were fitted by the Chebyshev method according to an exponential equation IBa = -I2 · exp(-t/tau 2- I1·exp(-t/tau 1) - NI where IBa is the total current, I2 the fast inactivating component (I2 = 0.137 µA, 34.6% of IBa), I1 the slow inactivating component (I1 = 0.241 µA, 60.8% of IBa), and NI the noninactivating component (NI = 0.018 µA, 4.5% of IBa). Time constants for current inactivation were tau 2 = 42.5 ms and tau 1 = 219.1 ms in this example. Data were fitted 12 ms after the start of depolarization. Data (open symbols) were shown after filtering 1/10. C, average tau 2 time constant as a function of depolarization value for alpha 1Abeta 4 and alpha 1Abeta 4Delta C-injected oocytes. Asterisks represent data statistically different from control (t test; p <=  0.1). D, average percentage of each inactivating component present in total current for alpha 1Abeta 4 and alpha 1Abeta 4Delta C-injected oocytes. Statistically significant results are shown by the asterisk (t test; p <=  0.1). E, modeled current inactivation based on average tau 2, tau 1, percent of I2, percent of I1, and percent of NI at 20 mV for both alpha 1Abeta 4 and alpha 1Abeta 4Delta C.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We describe here the identification of a new interaction site between the alpha 1A calcium channel subunit and the beta 4 subunit. The interaction is of low affinity compared with that between the AID site on the I-II cytoplasmic loop of alpha 1 subunits and the BID site of beta  subunits. This and the fact that mutations in AID or BID disrupt the interaction between the two subunits in expression experiments (17, 18) suggest that such an interaction would be of a secondary nature, dependent on formation of the initial AID-BID interaction to bring the interaction sites into close proximity, or to introduce conformational constraints that favor the interaction. The alternative possibility, that two beta  subunits would be associated to a single alpha 1 (e.g. as proposed by Tareilus et al. (20)), seems unlikely for several reasons. First, the molar ratio between alpha 1 and beta  subunits is 1:1 in purified channels (2, 23). Second, studies of the AID-BID interaction revealed that beta  subunits with affinities lower than 100 nM for AID do not associate with alpha 1 upon coexpression in oocytes (14), although, of course, it cannot be ruled out that in native conformation the affinity between the two carboxyl-terminal sequences is higher than that predicted from in vitro experience. Third, and most important, expression of beta  subunits containing disruptive BID mutations fail to modify channel properties, ruling out additional interactions in these conditions.

The carboxyl-terminal tails of alpha 1 subunits play various roles in channel function. In the alpha 1C subunit, deletion of the distal regions of the carboxyl terminus results in increased channel opening probability (24), and the more proximal EF-hand domain plays a role in Ca2+-induced inactivation (25). The carboxyl-terminal tail is also known to undergo post-translational modifications in the form of phosphorylation and proteolysis, in some cases essential to channel function (26). Interestingly, there are many phoshorylation sites present in both the alpha 1A carboxyl-terminal and beta 4 carboxyl-terminal sequence that may be of functional relevance. In beta 4 several of these phosphorylation sites are unique to this subunit. These data point to the functional importance of this region in channel regulation and may also provide the key to the main function of the subunit interaction site we describe here.

We show that, at least in vitro, beta 4 shows a much greater affinity for the carboxyl-terminal region of alpha 1A than does beta 2a, while no interaction is detected for beta 1b or beta 3. These differences in affinity suggest a functional significance that may help to explain the differences in functional effects seen for different combinations of alpha 1 and beta  subunits. In light of this, it is interesting that beta 4 is coexpressed in the same brain regions as is alpha 1A, particularly in the cerebellum (21, 27), and is the major beta  subunit associated with the alpha 1A subunit in the P/Q channel-type (16). A similar interaction has recently been reported between an alpha 1E splice variant and beta 2a (20). It will be interesting to see whether this interaction also displays a specificity for a particular subset of beta  subunits.

The advantage of a form of subunit specificity in the alpha 1-beta association remains largely to be investigated. Our data suggest that a secondary interaction site that favors beta 4 association to BI-2 rather than beta 3, the other predominant beta  in brain (28), could be determinant in underlying subtle kinetic differences induced by the various beta  subunits. Besides obvious functional differences, specific alpha 1-beta associations may be determinant in various aspects of channel biosynthesis such as channel targeting. Brice et al. (29) and Chien et al. (30) have indeed demonstrated that beta  subunits are crucial to cell surface localization of alpha 1.

Alternatively, it is possible that the carboxyl-terminal sequences of both subunits contribute to the process of channel clustering known to occur in voltage-dependent calcium channels, with the carboxyl-terminal sequence of the beta  subunit interacting with the carboxyl terminus of an alpha 1 subunit other than the one that it is attached to via its BID site. Channel clustering is known to occur in various ion channels and has best been characterized for the shaker K+ channel for which clustering is produced by the PSD-95 proteins (31). Calcium channel clustering is probably induced by a third party protein because the carboxyl-terminal interaction described herein may be of insufficient affinity to be the primary cause of such a clustering behavior.

Besides the existence of separate genes encoding Ca2+ channel subunits, alternative splicing is another process by which diversity can be introduced. The functional significance of alternative splicing in Ca2+ channel subunits is still largely unknown. Splicing can occur in several regions of the alpha 1 protein, including the amino terminus (32), the IS6 transmembrane sequence, the cytoplasmic linkers between domain I and II and between domain II and III (32), transmembrane segment IVS3, and the carboxyl-terminal sequences (21, 33). Particularly pertinent to the data presented here is the existence of two splice alternatives in alpha 1A described by Mori et al. (21) that result in an almost total divergence of sequence from residue 2230 onward, i.e. concerning the majority of the sequence responsible for the interaction studied here. It is therefore likely that the carboxyl terminus of the other alpha 1A splice variant, BI-1, may not interact with beta 4. Generally, it remains to be seen whether the splicing occurring in the carboxyl-terminal tail of the alpha 1 subunit plays a role in the specificity of the secondary alpha 1-beta interaction. Two case scenarios can be discussed. The first is that any deletion or insertion may modify the regulatory input of the associated beta  subunit at that location without modifying the type of beta  subunit associated. The second possibility is that splicing modifies the alpha 1-beta interaction specificity and that it favors the association of another type of beta  subunit, presumably to specify a different membrane targeting of the channel. In the case of alpha 1A, it would be interesting to see whether the carboxyl terminus of BI-1 interacts with beta 3, the other predominant beta  subunit known to interact with alpha 1A in the brain (16). Our data also shed new light on data obtained by other groups that report a lack of impact of alpha 1 carboxyl-terminal alternative splicing on beta  subunit regulation (for instance in alpha 1C (34), but see Soldatov et al. (35)) and suggest that negative data may well be due to the use of an inappropriate combination of alpha 1 and beta  subunits.

It is becoming increasingly obvious that a wide range of neurological and motor diseases result from mutations in the alpha 1 or beta  subunits, and a number of these are particularly relevant to the data we have presented here. In mice, the leaner phenotype, similar to absence epilepsy, has been attributed to a mutation in a splice donor consensus sequence of alpha 1A, resulting in aberrant splicing and therefore degeneration of the coding sequence corresponding to either residue 2026 or 2072 onward of the protein we have used (36), i.e. corresponding well to the region identified as interacting with beta 4. In humans, a severe form of ataxia has been shown to be associated with a 5-base pair insertion close to the stop codon that extends the translated sequence to include a glutamine repeat of variable size (37), which would presumably entail conformational changes to this region. Concerning the beta 4 subunit, its overall functional importance has been shown by the assignment of a lethargic phenotype in mice to a deletion of about 60% of the protein (38), although such a drastic alteration is likely to completely inactivate the beta 4 subunit and at least leads to the loss of the BID in addition to the carboxyl-terminal site.

Finally, the data obtained here contribute to our understanding of the general organization of high voltage-activated calcium channels. The existence of several sites of interaction between the two channel components highlights the utility of an in vitro approach using fusion proteins, since it enables us to study such interactions individually and therefore to assess their functional impact and to gradually dissect the conformational basis of the relationship between the two subunits. It is not always possible, however, to extrapolate directly between the situation in vitro and that in vivo. For example, our inability to demonstrate an effect of AID-beta association on the affinity of the interaction between the carboxyl termini of alpha 1A and beta 4 probably reflects the absence of the remainder of the alpha 1A molecule and therefore a loss of integrity of the conformational constraints existing in native channels which may determine the overall manner in which the two subunits interact.

    ACKNOWLEDGEMENTS

We thank Dr. H. Liu for the GST-2090-2424A construct and for helpful comments on the manuscript, Dr. V. Scott for the GST-1889-2126A construct, and Dr. R. Felix for reading the manuscript.

    FOOTNOTES

* 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 an INSERM postdoctoral fellowship (Poste Vert).

par An investigator of the Howard Hughes Medical Institute.

** To whom correspondence should be addressed: INSERM U464, Institut Fédératif Jean Roche, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France. Tel.: 33-4-91698860; Fax: 33-4-91090506; E-mail: dewaard.m{at}jean-roche.univ-mrs.fr.

1 The abbreviations used are: AID, alpha 1 subunit interaction domain; BID, beta  subunit interaction domain; PCR, polymerase chain reaction; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

2 M. De Waard, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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