A New beta  Subtype-specific Interaction in alpha 1A Subunit Controls P/Q-type Ca2+ Channel Activation*

Denise WalkerDagger , Delphine BichetDagger , Sandrine GeibDagger , Emiko Mori§, Véronique CornetDagger , Terry P. Snutch, Yasuo Mori§, and Michel De WaardDagger parallel

From Dagger  INSERM Unité 464, Institut Fédératif Jean Roche, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille cedex 20, France, the § Department of Information on Physiology, National Institute of Physiological Science, Okazaki 444, Aichi, Japan, and the  Biotechnology Laboratory, Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cytoplasmic beta  subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha 1 interaction domain (AID) in the I-II cytoplasmic loop of alpha 1 and the beta  interaction domain (BID) of the beta  subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha 1A (BI-2) and the carboxyl terminus of beta 4. Like the interaction identified previously (21) between the carboxyl termini of alpha 1A and beta 4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta 4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha 1 is isoform-dependent, suggesting a role in the specificity of alpha 1-beta pairing. Coexpression of beta 4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha 1A channel compared with beta 3 (not exhibiting this interaction). Replacing the amino terminus of alpha 1A with that of alpha 1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite their functional diversity, high voltage-gated Ca2+ channels have three subunit types in common (1, 2). The alpha 1, pore-forming component of the channel is associated with a cytoplasmic beta  subunit of 52-78 kDa and a largely extracellular alpha 2delta component, anchored by a single transmembrane domain. These subunits are encoded by at least 7 alpha 1, 4 beta , and 1 alpha 2delta genes, respectively, of which numerous splice variants exist (3).

The beta  subunit, when coexpressed with the alpha 1 subunit, results in an increase in current density, alteration of the voltage dependence and kinetics of both inactivation and activation, and an increase in the number of recognition sites for channel-specific ligands (for review, see Refs. 4 and 5). These effects reflect not only conformational modulation but also an increase in the number of channels properly addressed to the cell surface, suggesting multiple roles for the beta  subunit. Although the effects of beta  are highly conserved, significant differences are seen depending on the combination of alpha 1 and beta  subunits studied. For example, the kinetics of inactivation shows a general trend of variation with beta  subtype (6-9), whereas a shift in the voltage dependence of inactivation has been reported only for non-L-type, A, B, and E (10-12), and not L-type channels (13). beta  subunits also seem to differ in the mechanism by which they become localized to the plasma membrane (14, 15), perhaps suggesting that they are differentially targeted. Finally, alpha 1 and beta  subtypes differ in their potential (based on sequence predictions) to be phosphorylated by various protein kinases. These factors together point to a functional explanation for the growing evidence that the in vitro promiscuity of alpha 1-beta interactions is reflected by a heterogeneity of combinations in native channels (N (16), P/Q (17), and L type (18)).

Preliminary studies (10, 19) have identified a high affinity interaction between a highly conserved region in the cytoplasmic loop linking transmembrane regions I and II of alpha 1 (AID,1 or alpha 1 interaction domain) and a 30-residue region in the second conserved domain of beta  subunits (BID, or beta  interaction domain). This interaction occurs with a stoichiometry of 1:1 (20) and (at least in vitro and in expression systems) occurs between all combinations of alpha 1 and beta  subtypes tested so far. We have since reported (21) the existence of a subunit-specific interaction between the carboxyl-terminal domain of alpha 1A and the most carboxyl-terminal 109 residues of beta 4, and a similar interaction has been reported (22, 23) between alpha 1E and beta 2a. The comparative high affinity of the AID-BID interaction (20, 21), coupled with the abolition of all beta  modulatory effects by mutation of residues critical to the interaction between AID and BID (10, 19), suggests that this interaction represents a primary, anchoring interaction upon which further, secondary, interactions might depend. The specificity of such secondary interactions, or at least differences in affinity, represents a potential source for the variation seen for different alpha 1 and beta  combinations, in terms of both the electrophysiological properties of the channel and potential differences in control by other cellular factors, such as protein kinases and G proteins. We therefore set out to determine whether further secondary interaction sites exist. The present report describes the identification of an interaction between the amino-terminal cytoplasmic region of alpha 1A and the beta 4 subunit of P/Q channels providing a refreshed understanding of the molecular organization of voltage-dependent calcium channels. The interaction plays a critical role in the precise positioning of the channel activation process on the voltage axis. It constitutes yet another molecular determinant underlying functional differences among various beta  subunits and, by extension, probably among various native P/Q channel subtypes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GST Fusion Proteins-- Regions of the rabbit brain alpha 1A cDNA (BI-2 (24)) corresponding to residues 2-98 (i.e. the entire amino-terminal region), 2-52, 43-77, 66-98, and 76-98 were amplified by PCR and, with the aid of BamHI and EcoRI restriction sites included in the primers, were subcloned into pGEX2TK (Amersham Pharmacia Biotech). The resulting recombinant plasmids were expressed in Escherichia coli BL21, and the GST fusion proteins were purified as described previously (20). Fusion proteins expressing the entire amino-terminal regions (minus start codon) of alpha 1B (amino acids 2-95, GenBank M92905 (25)), alpha 1C (amino acids 2-151, M57974 (26)), and alpha 1S (amino acids 2-49, M23919 (27)) were constructed and purified similarly. The resulting fusion proteins are referred to as, for example, GST-NTA for that containing the entire amino-terminal region of alpha 1A and GST-NTA,2-52 for the truncated form of this which contains only residues 2-52.

In Vitro Translation of beta  Subunits-- beta 1b, beta 2a, beta 3, and beta 4 cDNA clones were as described previously (21). Truncated derivatives of beta 4 were constructed by PCR amplification of the corresponding regions of cDNA and subcloning into pcDNA3 (Invitrogen), using HindIII and BamHI sites (added to the PCR primers) with the addition of a Kozak sequence (28) and initiation codon (ACCATGG) or termination codon (TGA) as necessary. The beta 3/4 chimera construct (beta 3 1-360/beta 4 402-519 in pcDNA3) is as described previously (21). 35S-Labeled beta  subunits were synthesized in vitro using the TNTTM-coupled Transcription/Translation System (Promega). Non-incorporated [35S]methionine was removed by purification on a PD10 column (Amersham Pharmacia Biotech).

Binding Assays-- These were carried out using fusion proteins coupled to glutathione-agarose in Tris-buffered saline as described previously (21). Binding reactions were incubated for 5 h unless otherwise stated.

Peptides-- A 21-amino acid peptide containing the AIDA sequence QQQIERELNGYMEWISKAEEV and a 21-amino acid peptide containing residues 76-96 of the alpha 1A amino-terminus (RSLFLFSEDNVVRKYAKKITE) were synthesized by Genosys (United Kingdom).

Competition Experiments-- For competition experiments, a maltose-binding protein (MBP) in fusion with the carboxyl-terminal binding site of alpha 1A (amino acids 2120-2275) was constructed using the BamHI/SalI sites of pMAL-c2 (MBP-CTA,2120-2275). The effects of beta -AIDA and beta -CTA associations on beta -AIDA, beta -CTA, and beta -NTA interaction were analyzed by saturating each beta  site by preincubating the [35S]methionine-labeled beta 4 subunit (35S-beta 4) with 10 µM AIDA peptide (1 h) or 2 µM MBP-CTA,2120-2275 (4 h). Binding of 35S-beta 4 to various alpha 1A binding sites was then tested by a 4-h incubation with 250 nM GST (control), 250 nM GST-AIDA (AID site), 2 µM GST-NTA, and 2 µM GST-CTA,2090-2424, precipitation of glutathione-agarose beads, gel electrophoresis, and autoradiography.

Chimera alpha 1A Subunit-- Pairs of primers CSl-N1(+) 5'-GGGTCGACTAAAACGTAAAGTATTACTAAAACCTCAATTTGCAG-3' and BIC-N1(-) 5'-GTACTCAAAGGGTTTCCACTCGACGATGCT-3', and primers BIC-N1(+) 5'-GTCGAGTGGAAACCCTTTGAGTACATGATT-3' and BINt-N1(-) 5'-GAGCGGCCGCAGCACCCGCACTGC-3' were combined with the templates pCARD3 (29) and pSPBI-2 (24), respectively, in PCR amplification using the Advantage PCR kit (CLONTECH). The resulting PCR products and the primers CSl-N1(+) and BINt-N1(-) were subjected to subsequent PCR amplification to yield a chimeric sequence that contains nucleotides -191 to 462 from the alpha 1C sequence (29) and 336-650 from the alpha 1A sequence (24). The chimeric fragment was digested with SalI and NotI and ligated with the 11.8 kilobase NotI (partially digested)/SalI fragment from pSPBI-2 to yield pSP72C(N)-BI-2 (alpha 1A(NT)C subunit).

Electrophysiology-- Xenopus oocytes were prepared as described previously (6). Stage V and VI oocytes were injected with alpha 1A (BI-2 (24)) or alpha 1A(NT)C-specific mRNA (0.3 µg/µl) either alone or in combination with beta 3- or beta 4-specific mRNA (0.15 µg/µl) and maintained for 3-4 days before recording in defined nutrient oocyte medium (6). Two-electrode voltage clamp recording was performed at room temperature (18-20 °C) using 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, 0.5; pH 7.4 with methanesulfonic acid. Electrodes filled with 3 M KCl had a resistance of 0.1 megohm. Current records were filtered at 1 kHz, leak-subtracted on-line by a P/6 protocol, and sampled at 5 kHz. Residual capacitative currents were blanked. Data were analyzed using pCLAMP version 6.03 (Axon Instruments). All values are mean ± S.D.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A GST fusion protein, GST-NTA, expressing the entire amino-terminal cytoplasmic region of alpha 1A (splice variant BI-2) was assayed for in vitro binding to 35S-beta 4. As Fig. 1A shows, the NTA region exhibits a significant and specific interaction with beta 4 which is comparable to the binding observed to a GST fusion protein carrying the AIDA sequence. The binding of GST-NTA to 35S-beta 4 appears slightly stronger than the binding of GST-AIDA, but the relative efficiency of binding of these fusion proteins varied slightly depending on the beta -translation reactions used. The affinity of this interaction was determined by carrying out similar binding assays using a range of concentrations of GST-NTA fusion protein. Fig. 1B shows the resulting saturation curve, which is compared with that observed previously for the interaction of 35S-beta 4 with GST-AIDA. The affinity of interaction of GST-NTA is 100-fold lower (kD = 336 nM) than that for the AID interaction (close to 3 nM). These data are in favor of the idea that the AID-BID interaction represents a primary anchoring site of interaction between the two subunits which allows secondary interactions of lower affinity to occur. As already mentioned, it is interesting that in Fig. 1A, GST-NTA demonstrates greater binding than GST-AIDA to 35S-beta 4. Given that both fusion proteins are at concentrations giving maximal binding (Fig. 1B), this demonstrates a difference in maximal binding which appears to reflect a difference in conformational requirements, coupled with conformational heterogeneity in the 35S-beta 4 preparation (permissive and nonpermissive binding states; data not shown).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   In vitro binding of 35S-labeled beta 4 to the amino-terminal region of alpha 1A. Panel A. Left, Coomassie Blue-stained SDS-PAGE showing the GST fusion proteins used (5 µg). Right, autoradiogram of the binding assay. In vitro translated beta 4 was assayed for binding to the fusion proteins indicated (5 µM). GST, glutathione S-transferase alone; GST-AIDA, GST fused to AID region (residues 369-418) of alpha 1A; GST-NTA, GST fused to entire amino-terminal cytoplasmic domain (residues 2-98) of alpha 1A. After binding interactions as described under "Experimental Procedures," washed beads were analyzed by SDS-PAGE and autoradiography. Panel B, various concentrations of GST-NTA 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 concentration) and normalized by expression as a proportion of maximal binding. Error bars indicate normalized S.D. Data are described by a logistic function f = [(a - d)/(1 + (x/c)b] + d where a = 1. 019 and d = 0. 01022 are the asymptotic maximum and minimum, respectively; x is the fusion protein concentration; c = 336 nM is the Kd; and b = -2,816 is the slope of the curve. For comparison purposes, the saturation curve for AIDA-GST (Kd = 3 nM, dashed line) is also shown (20).

To characterize more precisely the region of alpha 1A responsible for interaction with beta 4, a series of GST fusion proteins carrying truncations of the region concerned (depicted in Fig. 2A) was constructed, and the proteins were assayed for their capacity to interact with 35S-beta 4. As Fig. 2B shows, removal of the most carboxyl-terminal amino acids, or of the 42 most amino-terminal, does not abolish the capacity to interact with beta 4. Concomitantly, fusion proteins corresponding to the most carboxyl-terminal region, which is most highly conserved among alpha 1 subtypes, are incapable of binding. In addition, the interaction between GST-NTA and 35S-beta 4 was not inhibited by addition to the binding reaction of a peptide (500 µM) corresponding to amino acids 76-98. These data suggest that the beta 4 binding site concerns a region between residues 1 and 66 of alpha 1A, maybe comprising, but not necessarily limited to, residues 42-52. The reduced binding to NTA,2-52 and NTA,42-77 compared with full-length NTA probably reflects instability and/or sequence reduction of the interaction site. The reduction in binding of smaller deleted derivatives meant that we were unable to pursue this approach further. A sequence alignment of this alpha 1A binding domain with equivalent domains of other alpha 1 subunits (alpha 1B, alpha 1E, alpha 1C, alpha 1D, and alpha 1S), some used in this investigation, suggests a relatively low level of sequence conservation, although alpha 1B and alpha 1A show some similarity (Fig. 2C). This observation implies that the interaction may not be conserved, a prediction that we went on to test (see Fig. 5).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Localization of the interaction site in alpha 1A. Panel A. Top, schematic diagram of the alpha 1A subunit. Amino acid positions are shown above, transmembrane domains (each composed of six membrane-spanning segments) are shown as dark boxes and numbered (I-IV) above. Bottom, enlargement of the amino-terminal domain, showing GST fusion proteins constructed with amino acid positions in alpha 1A marked at the extremities. Panel B. Top, Coomassie Blue-stained SDS-PAGE showing fusion proteins used (5 µg). Bottom, capacity of 5 µM purified fusion proteins to interact with 35S-beta 4. After the binding reactions, washed beads were analyzed by SDS-PAGE and autoradiography. Panel C, amino-terminal binding site of alpha 1A (BI-2, amino acids 1-66) and its alignment with sequences of other calcium channels (GenBank accession codes M92905, alpha 1B; X67855, alpha 1E; X15539, alpha 1C; M57682, alpha 1D; and M23919, alpha 1S).

To identify the region of beta 4 which interacts with the amino-terminal region of alpha 1A, we initially analyzed the binding capacity of several deleted derivatives of beta 4, translated in vitro (Fig. 3, A and B). These derivatives lacked either the amino-terminal, carboxyl-terminal, or both regions, which shows a low level of conservation among beta  subunit subtypes. As Fig. 3B shows, removal of the amino-terminal region had no effect, whereas removal of the carboxyl-terminal abolished binding completely, illustrating the importance of this region in the interaction. We also found that although beta 3 does not interact with GST-NTA (see Fig. 5), the opposite is true for a beta 3-beta 4 chimera, in which the nonconserved carboxyl terminus of beta 3 is replaced by the equivalent domain of beta 4 (Fig. 3B).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Localization of the interaction site in the beta 4 subunit. Panel A. Top, schematic map of beta 4 subunit, dividing the protein into five domains. Darker domains (II and IV) represent sequences of highest conservation among beta  subtypes (10). Bottom, autoradiography of SDS-PAGE showing beta 4 and deleted derivatives translated in vitro in the presence of [35S]methionine. Panel B, in vitro translated full-length, truncated beta 4 derivatives and a beta 3-beta 4 chimera (T) were assayed for their capacity to interact with GST and GST-NTA fusion proteins (2.5 µM) as described under "Experimental Procedures," and washed beads were analyzed by SDS-PAGE and autoradiography. Panel C, 35S-beta 4 and carboxyl-terminal deleted derivatives were assayed for their capacity to interact with 2.5 µM GST-NTA and a GST fusion protein containing residues 2090-2424 of alpha 1A (GST-CTA (21)). Specific binding was calculated by subtraction of binding to GST (at the same concentration) and normalized by expression as a percentage of maximal binding to GST-AIDA. Error bars represent normalized S.D.

We have shown previously (21) that the carboxyl-terminal region of beta 4 also interacts with the carboxyl-terminal cytoplasmic domain of alpha 1A (BI-2). We therefore wanted to map the two interaction sites more precisely, for which we constructed two additional derivatives of beta 4, lacking a third (residues 483-519) and two-thirds (residues 447-519) of the carboxyl terminus (Fig. 3, A and B). As Fig. 3C shows, deletion of residues 483-519 of beta 4 had no effect on its capacity to bind to GST-NTA, whereas truncation of the carboxyl terminus of beta 4 up to residue 446 resulted in a total loss of binding capacity. This indicates that the NTA binding region is located between residues 446 and 482 of beta 4. Analysis of the capacity of these truncates to bind to a GST fusion protein of the carboxyl-terminal region (residues 2090-2424) of alpha 1A (GST-CTA) resulted in binding capacity being gradually lost with each further deletion. This suggests that the binding site of CTA spans a wider region than the NTA binding site, is dependent on secondary or tertiary structures that are disrupted by the deletions, or consists of a series of dispersed sites. It is noteworthy that the previously characterized alpha 1A carboxyl-terminal binding site was also difficult to define, in that deleted derivatives over a long region retained binding capacity, giving support to the hypothesis that there are microdomains of interaction between these two sites (21). In contrast, the NTA site and corresponding domain on beta 4 are shorter and seem more easily delineated. In any case, the different patterns of interaction capacities seen for GST-NTA and GST-CTA suggest that these two regions of alpha 1A occupy different but overlapping sites on beta 4.

The involvement of overlapping regions of beta 4 in interactions with the amino- and carboxyl-terminal domains of alpha 1A also raised the question as to whether these interactions could occur simultaneously or whether they were mutually exclusive. To investigate this as well as their relationship with the AID-BID interaction, we tested whether the binding of AIDA (21-amino acid peptide) or GST-CTA to 35S-beta 4 could prevent its interaction with GST-NTA. The results, illustrated in Fig. 4, show that although the AID peptide was effective in preventing the interaction of beta 4 to GST-AIDA, it did not prevent the concomitant interaction with either GST-NTA or GST-CTA,2070-2275 (Fig. 4A). On the other hand, the association of MBP-CTA,2120-2275 with beta 4 blocked the ability of beta 4 to interact with GST-CTA,2070-2275 and also significantly reduced the binding of beta 4 to GST-NTA (Fig. 4B), suggesting that beta 4 is able to interact with AID and only one of the secondary interaction sites at a time.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   Multiple site occupancies on beta 4 subunit. Panel A, effect of 1-h AIDA peptide (10 µM) preincubation with 35S-beta 4 on control GST (250 nM), GST-AIDA (250 nM), GST-NTA (2 µM), and GST-CTA (2 µM) binding to 35S-beta 4. The 4-h binding reaction was conducted in the continued presence of AIDA peptide. Panel B, effect of 4-h MBP-CTA (2 µM) preincubation with 35S-beta 4 on control GST (250 nM), GST-NTA (2 µM), and GST-CTA (2 µM) binding to 35S-beta 4. The 4-h binding reaction was conducted in the continued presence of MBP-CTA. Complete inhibition of GST-NTA binding to 35S-beta 4 by MBP-CTA was difficult to achieve because MBP-CTA bound only a subset of the available 35S-beta 4, which was presumably in a more favorable conformation.

We have shown previously (21) that beta  subtypes differ in their capacity to interact with the carboxyl-terminal region of alpha 1A, with beta 4 interacting with greatest affinity, beta 2A with a lesser affinity, and beta 1b and beta 3 showing no significant interaction. We therefore wished to determine whether the same was true for interaction with the amino-terminal domain. In addition, because the beta  interaction site in the amino-terminal region of alpha 1A shows a variable level of conservation among alpha 1 subtypes, we wished to investigate whether beta  interaction capacities were conserved among them. Both of these questions were addressed by constructing a series of GST fusion proteins carrying the amino-terminal cytoplasmic region of alpha 1B, alpha 1C, and alpha 1S (Fig. 5A). These fusion proteins, along with GST alone, GST-AIDA (for comparison purposes), and GST-NTA, were assayed for their ability to interact with four different beta  subtypes, translated in vitro in presence of [35S]methionine (Fig. 5B). Interestingly, interaction with GST-NTA showed a pattern similar to that observed for the carboxyl-terminal region of alpha 1A (21) in that beta 4 exhibited the most significant interaction, beta 2a interacted to a lesser degree, and beta 1b and beta 3 showed no significant interaction. The amino-terminal domains of alpha 1B showed no significant interaction despite its closer sequence relatedness to alpha 1A. GST-NTS, on the other hand, showed significant interaction with all four beta  subunits, whereas GST-NTC, another L-type channel member, showed no interaction with any of the beta  subunits.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.   alpha 1 amino-terminal specificity of interaction with beta  subunits. Panel A, Coomassie Blue-stained SDS-PAGE showing various GST fusion proteins used (5 µg). Panel B, in vitro translated beta  subunits were assayed for their capacity to interact with 5 µM GST fusion proteins, and the remaining radioactivity associated with washed beads was quantified by counting. GST, control; AIDA, GST-AIDA; NTA, NTB, NTC, NTS, GST fusion proteins containing amino-terminal cytoplasmic domains of alpha 1A, alpha 1B, alpha 1C, and alpha 1S, respectively. Error bars represent S.D.

Because the amino-terminal sequences of alpha 1A and alpha 1S are very different, we checked whether binding of beta 4 to NTS involved the same interaction domain of beta 4. Fig. 6 demonstrates that, as for NTA, the carboxyl terminus of beta 4 was required for binding to NTS, and the use of deleted derivatives of the carboxyl terminus of beta 4 also indicates an important role for residues 446-482 of beta 4 in this interaction. These results suggest that the interaction site is defined more by the tertiary structure of the alpha 1 amino-terminal region than by its primary sequence, also explaining why the NTA site could not be localized more precisely than to residues 1-66 (Fig. 2).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   The carboxyl terminus of beta 4 is also involved in NTS binding. 35S-beta 4 and deleted derivatives were assayed for their capacity to interact with GST-NTS (5 µM). Specific binding was calculated by subtraction of binding to GST (at the same concentration) and normalized by expression as a percentage of maximal binding to GST-AIDA (500 nM). Error bars represent normalized S.D.

Finally, we questioned the relevance of the interaction between the amino terminus of alpha 1A and the carboxyl terminus of beta 4 in terms of channel functioning. First, because beta 3, in contrast to beta 4, does not interact with the amino terminus of alpha 1A, we investigated whether there were significant differences in terms of channel regulation by these two subunits. We found that in addition to triggering different inactivation kinetic behaviors (6), the two subunits differed in terms of their ability to shift the activation curve toward hyperpolarized potentials (Fig. 7A). Although both beta  subunits shifted the activation curve along the voltage axis, the shift induced by beta 3 was significantly more pronounced than the one produced by beta 4. The estimated half-activation potential shifted from 17 mV (alpha 1A-expressing oocytes) toward -13 mV (alpha 1Abeta 3 oocytes) and 1.5 mV (alpha 1Abeta 4 oocytes). There is thus an approximately 14-15 mV difference in the shift induced by the beta 3 and beta 4 subunits. In addition, we found that depending on the beta  subunit being expressed, the channels differed in their voltage dependence of inactivation with half-inactivation at -50 and -37 mV for alpha 1Abeta 3 and alpha 1Abeta 4 channels, respectively (data not shown). Because these differences in functional regulation by the various beta  subunits may be the result of differences in interaction levels between alpha 1A and the two beta  subunits, we determined the role of the NTA site in beta -induced channel regulation. We took advantage of the observation that essential differences were found in beta  subunit association with amino-terminal sequences of various alpha 1 subtypes. We constructed a chimera alpha 1A subunit (alpha 1A(NT)C), in which we replaced the amino terminus of alpha 1A (interacts with beta 4 but not beta 3) with the amino terminus of alpha 1C (does not interact with either beta 4 or beta 3). Coexpression of this chimeric channel with beta 3 or beta 4 triggers high voltage-activated currents in Xenopus oocytes (Fig. 7B). The amplitude of the currents elicited by membrane depolarization are reduced slightly compared with those obtained for the wild-type alpha 1A channel. Cells expressing alpha 1Abeta 3, for instance, have a peak current amplitude of 1,001 ± 651 nA (n = 7, S.D.), whereas cells expressing alpha 1A(NT)Cbeta 3 peak at 423 ± 655 nA (n = 12), which corresponds to a 2.37-fold reduction. A similar 2.06-fold reduction in current amplitude is seen when beta 4 is coexpressed with alpha 1A(NT)C (peak current 542 ± 240 nA, n = 12) rather than alpha 1A (peak current 1,118 ± 871 nA, n = 6). These results suggest that the amino terminus plays a role in channel expression levels at the plasma membrane but that beta  subunits and the NTA interaction site have little influence on this process. Also, the amino-terminal substitution induced an important shift in the voltage dependence of inactivation with half-inactivation occurring at -52 mV for alpha 1A(NT)Cbeta 4 channels compared with -37 mV for alpha 1Abeta 4 channels (data not shown). Because a similar shift is seen with beta 3 (not shown), this supports the idea that beta  subunit interaction with the amino terminus plays a minor role in this modification. In contrast, we found that the difference in the shift of voltage dependence of activation of alpha 1A(NT)Cbeta 3 and alpha 1A(NT)Cbeta 4 channels was reduced significantly (Fig. 7B). The average half-activation potential of alpha 1A(NT)Cbeta 3 channels was -13 mV and thus remained identical to that of the alpha 1Abeta 3 channels, whereas the V1/2 of alpha 1A(NT)Cbeta 4 channels was -9 mV, a significant hyperpolarizing shift compared with the alpha 1Abeta 4 channels. These data suggest that in the absence of an NTA/beta 4 interaction, the I-V shift induced by the beta 4 subunit resembles the shift induced by the beta 3 subunit. Finally, the substitution of the alpha 1A NTA sequence by NTC produced a slowing of channel inactivation with beta 4 but not with beta 3. The decay of alpha 1A(NT)Cbeta 4 currents occurred along two components with time constants of tau 1 = 80 ± 4 ms and tau 2 = 368 ± 39 ms (at 10 mV, n = 7) compared with tau 1 = 51 ± 9 ms and tau 2 = 246 ± 27 ms (n = 6) for alpha 1Abeta 4 currents. In contrast, no significant differences were seen in inactivation kinetics of alpha 1Abeta 3 or alpha 1A(NT)Cbeta 3 channels with time constants at 10 mV of tau 1 = 65 ± 15 ms and tau 2 = 243 ± 47 ms (n = 10) for alpha 1A(NT)Cbeta 3 currents and tau 1 = 62 ± 14 ms and tau 2 = 222 ± 13 ms (n = 7) for alpha 1Abeta 3 currents. These data further confirm a functional role in inactivation kinetics of the carboxyl terminus of beta 4 by its interaction with the carboxyl terminus (21) and amino terminus of the alpha 1A subunit.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 7.   Role of the NTA-beta 4 interaction in the control of voltage dependence of activation. A, beta 3 and beta 4 differ in their ability to shift the voltage dependence of activation of alpha 1A. Left and center, currents elicited by various membrane depolarizations (-30, -20, -10, and 0 mV) illustrating differences in threshold, intermediate, and peak activation of alpha 1Abeta 3 and alpha 1Abeta 4 channels expressed in Xenopus oocytes. Right, corresponding average current-voltage (I-V) relationship for alpha 1Abeta 3 (n = 7) and alpha 1Abeta 4 (n = 6) expressing cells. The I-V curve for cells expressing alpha 1A channel alone is shown for comparison purpose (n = 8). The experimental data were fitted with a modified Boltzmann equation IBa= (g·(V - E))/(1 + exp(-(V - V1/2)/k)), where g is the normalized conductance (g = 0.032, no beta ; 0.018, +beta 3; and 0.026, +beta 4); V1/2 is the half-activation potential (V1/2 = 17 mV, no beta ; -13 mV, +beta 3; and 1. 5 mV, +beta 4); E is the reversal potential (E = 67 mV, no beta ; 63 mV, +beta 3; and 58 mV, +beta 4); and k is the range of potential for an e-fold change around V1/2 (k = 7. 9 mV, no beta ; 4. 2 mV, +beta 3; and 5. 8 mV, +beta 4). B, change in difference in the beta -induced I-V shift by alpha 1A amino-terminal sequence substitution. Left and center, currents elicited by various membrane depolarizations (-30, -20, -10, and 0 mV) showing the absence of a difference in channel activation for alpha 1A(NT)Cbeta 3 and alpha 1A(NT)Cbeta 4 channels. Right, corresponding average I-V curves for alpha 1A(NT)Cbeta 3 (n = 13) and alpha 1A(NT)Cbeta 4 channels (n = 12). The fit of the experimental data yields V1/2 = -13 (+beta 3) and -9 mV (+beta 4); k = 4. 2 (+beta 3) and 4. 4 mV (+beta 4); g = 0. 017 (+beta 3) and 0. 018 (+beta 4); and E = 60 (+beta 3) and 63 mV (+beta 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We describe the identification of a specific interaction site between the amino-terminal cytoplasmic region of the calcium channel alpha 1A subunit and the beta 4 subunit. The beta 4 subunit is widely expressed in the brain, especially in the cerebellum (30). On the basis of their colocalization in many tissue types, beta 4 appears largely to be associated with the alpha 1A subunit in native channels (17, 31). However, coimmunoprecipitation studies demonstrate that alpha 1A is also found associated with beta 1b, beta 2, and beta 3 (17) and that beta 4 is also found associated with alpha 1B (16). The importance of the beta 4 subunit is illustrated by the recent demonstration that a lethargic phenotype in mice results from a deletion of approximately 60% of the beta 4 coding sequence (32). This truncated beta  subunit would lack all three of the interactions described with alpha 1A (AIDA (19) and NTA and CTA (21)), although such a deletion is also likely to result in severe conformational perturbation and probably degradation of the protein. This mutation is not entirely lethal, however, which is reminiscent of a growing number of experiments in which knockout of proteins of central importance does not turn out to be lethal. This is probably explained by a partial compensation by a related protein, in this case suggesting that other beta  subunits are expressed in parallel or that their expression is switched on to compensate for this deficiency (33). In fact, beta 3 is known to be a normal constituent of about one-third of P/Q-type channels (17). Because beta 3 expression is high in brain and parallels that of beta 4 (34), it would be the most likely candidate for beta 4 substitution in the lethargic mice. Since beta 3 lacks both secondary interaction sites described so far in beta 4, such a substitution would not be functionally equivalent, perhaps explaining some of the neurological defects encountered in these mice.

The NTA interaction identified is of relatively low affinity, supporting the idea that this is one of several secondary interactions between the two subunits that rely on the initial, high affinity interaction between the AID and BID sites identified previously. This idea is supported by the observation that mutagenesis of AID or BID to disrupt interaction between the two sites also disrupts the ability of the beta  subunit to modify channel properties (10). It also stems from the fact that this is the third interaction site mapped between alpha 1A and beta 4 and that binding of multiple beta  subunits to alpha 1 does not seem very plausible. The new interaction site that we describe involves the amino terminus of alpha 1A (residues 1-66) and carboxyl terminus (residues 446-482) of beta 4. This is particularly interesting given the rather low level of sequence conservation in the two regions identified. With regard to alpha 1A splice variants, the sequence of the amino-terminal cytoplasmic region is identical in BI-1 and BI-2 subtypes, indicating conservation of this interaction (24). This is in contrast to the beta 4 interaction site that we have identified previously in the carboxyl-terminal region of the BI-2 splice variant.

The low degree of sequence conservation observed for the respective interaction sites identified in the amino-terminal region of alpha 1A and the carboxyl-terminal of beta 4 is reflected by the high degree of subtype specificity exhibited by this interaction with respect to both alpha 1 and beta  isoforms. Our results indicate that the equivalent amino-terminal regions of alpha 1B and alpha 1C did not interact with any of the beta  subunits tested. Because other beta  subtypes exist, we cannot rule out that this may reflect the use of an inappropriate alpha 1-beta combination. Interestingly, we found that a fusion protein expressing the entire amino terminus of alpha 1S could interact with all four different beta  subunits tested. This is in contrast to the NTA binding, which occurs only on beta 4 and to a lesser extent on beta 2a. These results are indicative of a potential interaction of alpha 1S with beta  subunits other than beta 1a, the major beta  subunit of skeletal muscle, and parallel recent findings that beta 3 (7, 32) and a beta 1 splice variant other than beta 1a (2) are also expressed, albeit at low levels, in skeletal muscle. Overall, our results are indicative of evolution to provide for alpha 1-beta interaction specificity both within the alpha 1 amino-terminal and the beta  carboxyl-terminal sequences.

Fig. 8 summarizes what is now known about alpha 1A-beta 4 interactions in terms of structure. One interesting aspect is that the beta 4 subunit can interact simultaneously with AID and, via its carboxyl-terminal region, with either the amino- or carboxyl-terminal regions of alpha 1A, thereby defining two patterns of interactions. These interactions probably impose conformational constraints on the molecule which appear to affect channel function. It is also tempting to speculate that the conformational constraints are different depending on the patterns of interaction in use by the channel. The importance of the amino and carboxyl termini of alpha 1A are underlined by the observation that truncations of equivalent domains in alpha 1C result in enhanced current levels of the channel (35, 36). These enhanced current levels occur either by a greater membrane incorporation (amino terminus) or enhanced open probability (carboxyl terminus). Because beta  subunits also increase channel expression, and this effect varies in amplitude depending on the alpha 1 and beta  subtype studied, it is tempting to speculate that the secondary interaction sites described so far also intervene in alpha 1A channel expression by one of the two mechanisms described for alpha 1C. We did indeed find that substitution of the amino terminus of alpha 1A by the equivalent sequence of alpha 1C resulted in an important reduction in current density. This effect was, however, beta  subtype-independent, and it is therefore unlikely that the NTA interaction site described here plays a role in beta -induced enhancement of current amplitude. Despite this, secondary interactions appear to play other roles in several aspects of control of channel activity. We have shown previously (21) the importance of the carboxyl-terminal region of alpha 1A in the control of channel inactivation kinetics. Here, we demonstrate that the amino-terminal interaction site of alpha 1A is required for fine tuning the voltage dependence of activation. The NTA interaction with beta 4 appears to limit the amplitude of the hyperpolarizing beta -induced shift of channel activation. By this unique mechanism, it can be predicted that the beta 3-containing P/Q channel subtype is activated more easily than the beta 4-containing P/Q channel subtype. In addition, secondary interaction sites may serve to protect or uncover phosphorylation sites in the alpha 1A subunit, thereby altering the regulatory input of these. Another obvious possibility is that they play a role in the antagonistic relationship between the beta  subunit and Gbeta gamma complex. In this respect, it is interesting that Qin et al. (23) have recently shown that, in addition to interacting with a region overlapping with the AID site (37, 38), Gbeta gamma also interacts with the carboxyl-terminal domain of alpha 1A, alpha 1B, and alpha 1E and that the amino terminus has recently been recognized as another determinant for Gbeta gamma regulation in alpha 1E subunits (39). Finally, the existence of secondary interactions in addition to the AID-BID interaction could serve to favor certain combinations of subunits in cells where several subtypes are expressed. Given that beta  subunits also play a role in the surface targeting of alpha 1 and alpha 2delta (14), an interesting possibility is that specific beta  subunits serve to target alpha 1 subunits to specific regions of the cell surface.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 8.   Schematic alpha 1A-beta 4 interactions. Shown are the AID-BID anchoring site (As) implicating the I-II loop of alpha 1A and the first 30 residues of domain IV of beta 4; and the two secondary interactions sites, the first comprising NTA (Ss1) and the second CTA (Ss2), involving overlapping regions of domain V of beta 4.


    FOOTNOTES

* This work was supported by an INSERM postdoctoral fellowship (Poste Vert) (to D. W.).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.

parallel 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.: 33-4-9169-8860; Fax: 33-4-9109-0506; E-mail: dewaard.m{at}jean-roche.univ-mrs.fr.

    ABBREVIATIONS

The abbreviations used are: AID, alpha 1 interaction domain; BID, beta interaction domain; GST, glutathione S-transferase; PCR, polymerase chain reaction; MBP, maltose-binding protein; 35S-beta 4, [35S]methionine-labeled beta 4 subunit; PAGE, polyacrylamide gel electrophoresis. Fusion proteins are referred to as, for example, GST-NTA for that containing the entire amino-terminal region of alpha 1A, and GST-NTA,2-52 for the truncated form of this which contains only residues 2-52.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Flockerzi, V., Oeken, H.-J., Hofmann, F., Pelzer, D., Cavalie, A., and Trautwein, W. (1986) Nature 323, 66-68[Medline] [Order article via Infotrieve]
  2. Witcher, D. R., De Waard, M., Sakamoto, J., Franzini-Armstrong, C., Pragnell, M., Kahl, S. D., and Campbell, K. P. (1993) Science 261, 486-489[Medline] [Order article via Infotrieve]
  3. Birnbaumer, L., Campbell, K. P., Catterall, W. A., Harpold, M. M., Hofmann, F., Horne, W. A., Mori, Y., Schwartz, A., Snutch, T. P., Tanabe, T., and Tsien, R. W. (1994) Neuron 13, 505-506[Medline] [Order article via Infotrieve]
  4. Catterall, W. A. (1995) Annu. Rev. Biochem. 64, 493-531[CrossRef][Medline] [Order article via Infotrieve]
  5. Walker, D., and De Waard, M. (1998) Trends Neurosci. 21, 148-154[CrossRef][Medline] [Order article via Infotrieve]
  6. De Waard, M., and Campbell, K. P. (1995) J. Physiol. (Lond.) 485, 619-634[Abstract]
  7. Hullin, R., Singer-Lahat, D., Freichel, M., Biel, M., Dascal, N., Hofmann, F., and Flockerzi, V. (1992) EMBO J. 11, 885-890[Abstract]
  8. Perez-Reyes, E., Castellano, A., Kim, H. S., Bertrand, P., Baggstrom, E., Lacerda, A. E., Wei, X. Y., and Birnbaumer, L. (1992) J. Biol. Chem. 267, 1792-1797[Abstract/Free Full Text]
  9. Castellano, A., Wei, X. Y., Birnbaumer, L., and Perez-Reyes, E. (1993) J. Biol. Chem. 268, 12359-12366[Abstract/Free Full Text]
  10. De Waard, M., Pragnell, M., and Campbell, K. P. (1994) Neuron 13, 495-503[Medline] [Order article via Infotrieve]
  11. Soong, T. W., Stea, A., Hodson, C. D., Dubel, S. J., Vincent, S. R., and Snutch, T. P. (1993) Science 260, 1133-1136[Medline] [Order article via Infotrieve]
  12. Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., and Dascal, N. (1991) Science 253, 1553-1557[Medline] [Order article via Infotrieve]
  13. Tomlinson, W. J., Stea, A., Bourinet, E., Charnet, P., Nargeot, J., and Snutch, T. P. (1993) Neuropharmacology 32, 1117-1126[CrossRef][Medline] [Order article via Infotrieve]
  14. Brice, N. L., Berrow, N. S., Campbell, V., Page, K. M., Brickley, K., Tedder, I., and Dolphin, A. C. (1997) Eur. J. Neurosci. 9, 749-759[Medline] [Order article via Infotrieve]
  15. Chien, A. J., Zhao, X., Shirokov, R. E., Puri, T. S., Chang, C. F., Sun, D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036-30044[Abstract/Free Full Text]
  16. Scott, V. E. S., De Waard, M., Liu, H., Gurnett, C. A., Venzke, D. P., Lennon, V. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 3207-3212[Abstract/Free Full Text]
  17. Liu, H., De Waard, M., Scott, V. E. S., Gurnett, C. A., Lennon, V. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 13804-13810[Abstract/Free Full Text]
  18. Pichler, M., Cassidy, T. N., Reimer, D., Haase, H., Kraus, R., Ostler, D., and Striessnig, J. (1997) J. Biol. Chem. 272, 13877-13882[Abstract/Free Full Text]
  19. Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, T. P., and Campbell, K. P. (1994) Nature 368, 67-70[CrossRef][Medline] [Order article via Infotrieve]
  20. De Waard, M., Witcher, D. R., Pragnell, M., Liu, H., and Campbell, K. P. (1995) J. Biol. Chem. 270, 12056-12064[Abstract/Free Full Text]
  21. Walker, D., Bichet, D., Campbell, K. P., and De Waard, M. (1998) J. Biol. Chem. 273, 2361-2367[Abstract/Free Full Text]
  22. Tareilus, E., Roux, M., Qin, N., Olcese, R., Zhou, J., Stefani, E., and Birnbaumer, L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1703-1708[Abstract/Free Full Text]
  23. Qin, N., Platano, D., Olcese, R., Stefani, E., and Birnbaumer, L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8866-8871[Abstract/Free Full Text]
  24. Mori, Y., Friedrich, T., Kim, M.-S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., Mikoshiba, K., Imoto, K., Tanabe, T., and Numa, S. (1991) Nature 350, 398-402[CrossRef][Medline] [Order article via Infotrieve]
  25. Dubel, S. J., Starr, T. V., Hell, J., Ahlijanian, M. K., Enyeart, J. J., Catterall, W. A., and Snutch, T. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5058-5062[Abstract]
  26. Perez-Reyes, E., Wei, X. Y., Castellano, A., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 20430-20436[Abstract/Free Full Text]
  27. Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kohima, M., Matsuo, H., Hirose, T., and Numa, S. (1987) Nature 328, 313-318[CrossRef][Medline] [Order article via Infotrieve]
  28. Kozak, M. (1986) Cell 44, 283-292[Medline] [Order article via Infotrieve]
  29. Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989) Nature 340, 230-233[CrossRef][Medline] [Order article via Infotrieve]
  30. Tanaka, O., Sakagami, H., and Kondo, H. (1995) Mol. Brain Res. 30, 1-16[CrossRef][Medline] [Order article via Infotrieve]
  31. Ludwig, A., Flockerzi, V., and Hofmann, F. (1997) J. Neurosci. 17, 1339-1349[Abstract/Free Full Text]
  32. Burgess, D. L., Jones, J. M., Meisler, M. H., and Noebels, J. L. (1997) Cell 88, 385-392[Medline] [Order article via Infotrieve]
  33. McEnery, M. W., Copeland, T. D., and Vance, C. L. (1998) Soc. Neurosci. Abstr. 24, 81
  34. Witcher, D. R., De Waard, M., Liu, H., Pragnell, M., and Campbell, K. P. (1995) J. Biol. Chem. 270, 18088-18093[Abstract/Free Full Text]
  35. Wei, X., Neely, A., Lacerda, A. E., Olcese, R., Stefani, E., Perez-Reyes, E., and Birnbaumer, L. (1994) J. Biol. Chem. 269, 1635-1640[Abstract/Free Full Text]
  36. Wei, X., Neely, A., Olcese, R., Lang, W., Stefani, E., and Birnbaumer, L. (1996) Recept. Channels 4, 205-215[Medline] [Order article via Infotrieve]
  37. De Waard, M., Liu, H., Walker, D., Scott, V. E. S., Gurnett, C. A., and Campbell, K. P. (1997) Nature 385, 446-450[CrossRef][Medline] [Order article via Infotrieve]
  38. Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446[CrossRef][Medline] [Order article via Infotrieve]
  39. Page, K. M., Canti, C., Stephens, G. J., Berrow, N. S., and Dolphin, A. C. (1998) J. Neurosci 18, 4815-4824[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.