A New
Subtype-specific Interaction in
1A
Subunit Controls P/Q-type Ca2+ Channel Activation*
Denise
Walker
,
Delphine
Bichet
,
Sandrine
Geib
,
Emiko
Mori§,
Véronique
Cornet
,
Terry P.
Snutch¶,
Yasuo
Mori§, and
Michel
De Waard
From
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 |
The cytoplasmic
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
1 interaction domain (AID) in the I-II
cytoplasmic loop of
1 and the
interaction domain
(BID) of the
subunit is highly conserved among subunit subtypes. We
describe a new subtype-specific interaction (Ss1) between the
amino-terminal cytoplasmic domain of
1A (BI-2) and the
carboxyl terminus of
4. Like the interaction identified
previously (21) between the carboxyl termini of
1A and
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
4 and are competitive, but
neither inhibits the interaction with AID. The interaction with the
amino terminus of
1 is isoform-dependent, suggesting a role in the specificity of
1-
pairing.
Coexpression of
4 in Xenopus oocytes
produces a reduced hyperpolarizing shift in the I-V curve
of the
1A channel compared with
3 (not
exhibiting this interaction). Replacing the amino terminus of
1A with that of
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 |
Despite their functional diversity, high voltage-gated
Ca2+ channels have three subunit types in common (1, 2).
The
1, pore-forming component of the channel is
associated with a cytoplasmic
subunit of 52-78 kDa and a largely
extracellular
2
component, anchored by a single
transmembrane domain. These subunits are encoded by at least 7
1, 4
, and 1
2
genes, respectively, of which numerous splice variants exist (3).
The
subunit, when coexpressed with the
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
subunit. Although the effects of
are highly
conserved, significant differences are seen depending on the
combination of
1 and
subunits studied. For example,
the kinetics of inactivation shows a general trend of variation with
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).
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,
1 and
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
1-
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
1
(AID,1 or
1
interaction domain) and a 30-residue region in the second conserved
domain of
subunits (BID, or
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
1 and
subtypes tested so far. We
have since reported (21) the existence of a subunit-specific
interaction between the carboxyl-terminal domain of
1A
and the most carboxyl-terminal 109 residues of
4, and a
similar interaction has been reported (22, 23) between
1E and
2a. The comparative high affinity of the AID-BID interaction (20, 21), coupled with the abolition of all
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
1 and
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
1A and the
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
subunits and, by extension, probably
among various native P/Q channel subtypes.
 |
EXPERIMENTAL PROCEDURES |
GST Fusion Proteins--
Regions of the rabbit brain
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
1B (amino acids 2-95,
GenBank M92905 (25)),
1C (amino acids 2-151, M57974
(26)), and
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
1A and
GST-NTA,2-52 for the truncated form of this which contains
only residues 2-52.
In Vitro Translation of
Subunits--
1b,
2a,
3, and
4 cDNA
clones were as described previously (21). Truncated derivatives of
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
3/4 chimera construct (
3
1-360/
4 402-519 in pcDNA3) is as described
previously (21). 35S-Labeled
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
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
1A (amino acids 2120-2275) was
constructed using the BamHI/SalI sites of pMAL-c2
(MBP-CTA,2120-2275). The effects of
-AIDA
and
-CTA associations on
-AIDA,
-CTA, and
-NTA interaction were analyzed by saturating each
site by preincubating the
[35S]methionine-labeled
4 subunit
(35S-
4) with 10 µM
AIDA peptide (1 h) or 2 µM
MBP-CTA,2120-2275 (4 h). Binding of
35S-
4 to various
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
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
1C sequence (29) and
336-650 from the
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
(
1A(NT)C subunit).
Electrophysiology--
Xenopus oocytes were prepared
as described previously (6). Stage V and VI oocytes were injected with
1A (BI-2 (24)) or
1A(NT)C-specific mRNA (0.3 µg/µl)
either alone or in combination with
3- or
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 |
A GST fusion protein, GST-NTA, expressing the entire
amino-terminal cytoplasmic region of
1A (splice variant
BI-2) was assayed for in vitro binding to
35S-
4. As Fig.
1A shows, the NTA
region exhibits a significant and specific interaction with
4 which is comparable to the binding observed to a GST
fusion protein carrying the AIDA sequence. The binding of
GST-NTA to 35S-
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
-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-
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-
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-
4 preparation
(permissive and nonpermissive binding states; data not shown).

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Fig. 1.
In vitro binding of
35S-labeled 4 to the
amino-terminal region of 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
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 1A;
GST-NTA, GST fused to entire amino-terminal
cytoplasmic domain (residues 2-98) of 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- 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
1A
responsible for interaction with
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-
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
4. Concomitantly, fusion proteins corresponding to the
most carboxyl-terminal region, which is most highly conserved among
1 subtypes, are incapable of binding. In addition, the
interaction between GST-NTA and
35S-
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
4
binding site concerns a region between residues 1 and 66 of
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
1A binding domain with
equivalent domains of other
1 subunits
(
1B,
1E,
1C,
1D, and
1S), some used in this
investigation, suggests a relatively low level of sequence
conservation, although
1B and
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).

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Fig. 2.
Localization of the interaction site in
1A. Panel A. Top, schematic diagram of the 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 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- 4. After the
binding reactions, washed beads were analyzed by SDS-PAGE and
autoradiography. Panel C, amino-terminal binding site of
1A (BI-2, amino acids 1-66) and its alignment with
sequences of other calcium channels (GenBank accession codes M92905,
1B; X67855, 1E; X15539,
1C; M57682, 1D; and M23919,
1S).
|
|
To identify the region of
4 which interacts with the
amino-terminal region of
1A, we initially analyzed the
binding capacity of several deleted derivatives of
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
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
3 does not interact with
GST-NTA (see Fig. 5), the opposite is true for a
3-
4 chimera, in which the nonconserved carboxyl terminus of
3 is replaced by the equivalent
domain of
4 (Fig. 3B).

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Fig. 3.
Localization of the interaction site in
the 4 subunit. Panel
A. Top, schematic map of 4 subunit,
dividing the protein into five domains. Darker domains (II
and IV) represent sequences of highest conservation among subtypes
(10). Bottom, autoradiography of SDS-PAGE showing
4 and deleted derivatives translated in vitro
in the presence of [35S]methionine. Panel B,
in vitro translated full-length, truncated 4
derivatives and a 3- 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- 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 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.
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|
We have shown previously (21) that the carboxyl-terminal region of
4 also interacts with the carboxyl-terminal cytoplasmic domain of
1A (BI-2). We therefore wanted to map the two
interaction sites more precisely, for which we constructed two
additional derivatives of
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
4 had no effect on its
capacity to bind to GST-NTA, whereas truncation of the carboxyl terminus of
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
4. Analysis of the capacity of these truncates to bind
to a GST fusion protein of the carboxyl-terminal region (residues
2090-2424) of
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
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
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
1A occupy different but overlapping sites on
4.
The involvement of overlapping regions of
4 in
interactions with the amino- and carboxyl-terminal domains of
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-
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
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
4 blocked the
ability of
4 to interact with
GST-CTA,2070-2275 and also significantly reduced the
binding of
4 to GST-NTA (Fig.
4B), suggesting that
4 is able to interact
with AID and only one of the secondary interaction sites at a time.

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Fig. 4.
Multiple site occupancies on
4 subunit. Panel A,
effect of 1-h AIDA peptide (10 µM)
preincubation with 35S- 4 on control GST (250 nM), GST-AIDA (250 nM),
GST-NTA (2 µM), and GST-CTA (2 µM) binding to 35S- 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- 4 on control GST (250 nM),
GST-NTA (2 µM), and GST-CTA (2 µM) binding to 35S- 4. The 4-h
binding reaction was conducted in the continued presence of
MBP-CTA. Complete inhibition of GST-NTA binding
to 35S- 4 by MBP-CTA was
difficult to achieve because MBP-CTA bound only a subset of
the available 35S- 4, which was presumably in
a more favorable conformation.
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We have shown previously (21) that
subtypes differ in their
capacity to interact with the carboxyl-terminal region of
1A, with
4 interacting with greatest
affinity,
2A with a lesser affinity, and
1b and
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
interaction site in the amino-terminal region of
1A shows a variable level of conservation among
1 subtypes, we wished to investigate whether
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
1B,
1C, and
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
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
1A (21) in that
4 exhibited the most
significant interaction,
2a interacted to a lesser
degree, and
1b and
3 showed no
significant interaction. The amino-terminal domains of
1B showed no significant interaction despite its closer
sequence relatedness to
1A. GST-NTS, on the
other hand, showed significant interaction with all four
subunits,
whereas GST-NTC, another L-type channel member, showed no
interaction with any of the
subunits.

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Fig. 5.
1 amino-terminal
specificity of interaction with subunits. Panel A, Coomassie Blue-stained
SDS-PAGE showing various GST fusion proteins used (5 µg). Panel
B, in vitro translated 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
1A, 1B, 1C, and
1S, respectively. Error bars represent
S.D.
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Because the amino-terminal sequences of
1A and
1S are very different, we checked whether binding of
4 to NTS involved the same interaction
domain of
4. Fig. 6
demonstrates that, as for NTA, the carboxyl terminus of
4 was required for binding to NTS, and the
use of deleted derivatives of the carboxyl terminus of
4
also indicates an important role for residues 446-482 of
4 in this interaction. These results suggest that the
interaction site is defined more by the tertiary structure of the
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).

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Fig. 6.
The carboxyl terminus of
4 is also involved in NTS
binding. 35S- 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.
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Finally, we questioned the relevance of the interaction between the
amino terminus of
1A and the carboxyl terminus of
4 in terms of channel functioning. First, because
3, in contrast to
4, does not interact
with the amino terminus of
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
subunits shifted the activation curve along the voltage axis, the shift induced by
3 was significantly more pronounced
than the one produced by
4. The estimated
half-activation potential shifted from 17 mV
(
1A-expressing oocytes) toward
13 mV
(
1A
3 oocytes) and 1.5 mV
(
1A
4 oocytes). There is thus an
approximately 14-15 mV difference in the shift induced by the
3 and
4 subunits. In addition, we found
that depending on the
subunit being expressed, the channels
differed in their voltage dependence of inactivation with
half-inactivation at
50 and
37 mV for
1A
3 and
1A
4
channels, respectively (data not shown). Because these differences in
functional regulation by the various
subunits may be the result of
differences in interaction levels between
1A and the two
subunits, we determined the role of the NTA site in
-induced channel regulation. We took advantage of the observation
that essential differences were found in
subunit association with
amino-terminal sequences of various
1 subtypes. We
constructed a chimera
1A subunit
(
1A(NT)C), in which we replaced the amino
terminus of
1A (interacts with
4 but not
3) with the amino terminus of
1C (does
not interact with either
4 or
3).
Coexpression of this chimeric channel with
3 or
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
1A
channel. Cells expressing
1A
3, for
instance, have a peak current amplitude of 1,001 ± 651 nA
(n = 7, S.D.), whereas cells expressing
1A(NT)C
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
4 is coexpressed with
1A(NT)C (peak current 542 ± 240 nA,
n = 12) rather than
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
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
1A(NT)C
4 channels
compared with
37 mV for
1A
4 channels
(data not shown). Because a similar shift is seen with
3
(not shown), this supports the idea that
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
1A(NT)C
3 and
1A(NT)C
4 channels was reduced significantly (Fig. 7B). The average half-activation
potential of
1A(NT)C
3
channels was
13 mV and thus remained identical to that of the
1A
3 channels, whereas the
V1/2 of
1A(NT)C
4 channels was
9 mV,
a significant hyperpolarizing shift compared with the
1A
4 channels. These data suggest that in
the absence of an NTA/
4 interaction, the
I-V shift induced by the
4 subunit resembles
the shift induced by the
3 subunit. Finally, the
substitution of the
1A NTA sequence by
NTC produced a slowing of channel inactivation with
4 but not with
3. The decay of
1A(NT)C
4 currents occurred along two components with time constants of
1 = 80 ± 4 ms and
2 = 368 ± 39 ms (at 10 mV,
n = 7) compared with
1 = 51 ± 9 ms and
2 = 246 ± 27 ms (n = 6) for
1A
4 currents. In contrast, no significant
differences were seen in inactivation kinetics of
1A
3 or
1A(NT)C
3 channels with time
constants at 10 mV of
1 = 65 ± 15 ms and
2 = 243 ± 47 ms (n = 10) for
1A(NT)C
3 currents and
1 = 62 ± 14 ms and
2 = 222 ± 13 ms (n = 7) for
1A
3
currents. These data further confirm a functional role in inactivation
kinetics of the carboxyl terminus of
4 by its
interaction with the carboxyl terminus (21) and amino terminus of the
1A subunit.

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Fig. 7.
Role of the
NTA- 4 interaction in
the control of voltage dependence of activation. A,
3 and 4 differ in their ability to shift
the voltage dependence of activation of 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
1A 3 and 1A 4
channels expressed in Xenopus oocytes. Right,
corresponding average current-voltage (I-V) relationship for
1A 3 (n = 7) and
1A 4 (n = 6) expressing
cells. The I-V curve for cells expressing 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 ; 0.018, + 3; and 0.026, + 4);
V1/2 is the half-activation potential
(V1/2 = 17 mV, no ; 13 mV,
+ 3; and 1. 5 mV, + 4); E is the
reversal potential (E = 67 mV, no ; 63 mV,
+ 3; and 58 mV, + 4); and k is
the range of potential for an e-fold change around
V1/2 (k = 7. 9 mV, no ; 4. 2 mV, + 3; and 5. 8 mV, + 4). B,
change in difference in the -induced I-V shift by
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
1A(NT)C 3 and
1A(NT)C 4 channels.
Right, corresponding average I-V curves for
1A(NT)C 3 (n = 13) and 1A(NT)C 4 channels
(n = 12). The fit of the experimental data yields
V1/2 = 13 (+ 3) and 9 mV
(+ 4); k = 4. 2 (+ 3) and
4. 4 mV (+ 4); g = 0. 017 (+ 3) and 0. 018 (+ 4); and
E = 60 (+ 3) and 63 mV
(+ 4).
|
|
 |
DISCUSSION |
We describe the identification of a specific interaction site
between the amino-terminal cytoplasmic region of the calcium channel
1A subunit and the
4 subunit. The
4 subunit is widely expressed in the brain, especially
in the cerebellum (30). On the basis of their colocalization in many
tissue types,
4 appears largely to be associated with
the
1A subunit in native channels (17, 31). However,
coimmunoprecipitation studies demonstrate that
1A is
also found associated with
1b,
2, and
3 (17) and that
4 is also found
associated with
1B (16). The importance of the
4 subunit is illustrated by the recent demonstration
that a lethargic phenotype in mice results from a deletion of
approximately 60% of the
4 coding sequence (32). This
truncated
subunit would lack all three of the interactions
described with
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
subunits are expressed in parallel or that their expression is
switched on to compensate for this deficiency (33). In fact,
3 is known to be a normal constituent of about one-third
of P/Q-type channels (17). Because
3 expression is high
in brain and parallels that of
4 (34), it would be the
most likely candidate for
4 substitution in the
lethargic mice. Since
3 lacks both secondary interaction sites described so far in
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
subunit to modify channel properties
(10). It also stems from the fact that this is the third interaction site mapped between
1A and
4 and that
binding of multiple
subunits to
1 does not seem very
plausible. The new interaction site that we describe involves the amino
terminus of
1A (residues 1-66) and carboxyl terminus
(residues 446-482) of
4. This is particularly
interesting given the rather low level of sequence conservation in the
two regions identified. With regard to
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
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
1A and the carboxyl-terminal of
4 is
reflected by the high degree of subtype specificity exhibited by this
interaction with respect to both
1 and
isoforms. Our
results indicate that the equivalent amino-terminal regions of
1B and
1C did not interact with any of
the
subunits tested. Because other
subtypes exist, we cannot
rule out that this may reflect the use of an inappropriate
1-
combination. Interestingly, we found that a fusion
protein expressing the entire amino terminus of
1S could
interact with all four different
subunits tested. This is in
contrast to the NTA binding, which occurs only on
4 and to a lesser extent on
2a. These
results are indicative of a potential interaction of
1S
with
subunits other than
1a, the major
subunit
of skeletal muscle, and parallel recent findings that
3
(7, 32) and a
1 splice variant other than
1a (2) are also expressed, albeit at low levels, in
skeletal muscle. Overall, our results are indicative of evolution to
provide for
1-
interaction specificity both within
the
1 amino-terminal and the
carboxyl-terminal sequences.
Fig. 8 summarizes what is now known about
1A-
4 interactions in terms of structure.
One interesting aspect is that the
4 subunit can
interact simultaneously with AID and, via its carboxyl-terminal region,
with either the amino- or carboxyl-terminal regions of
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
1A are underlined by
the observation that truncations of equivalent domains in
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
subunits also increase channel expression, and
this effect varies in amplitude depending on the
1 and
subtype studied, it is tempting to speculate that the secondary
interaction sites described so far also intervene in
1A
channel expression by one of the two mechanisms described for
1C. We did indeed find that substitution of the amino
terminus of
1A by the equivalent sequence of
1C resulted in an important reduction in current
density. This effect was, however,
subtype-independent, and it is
therefore unlikely that the NTA interaction site described
here plays a role in
-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
1A in the control of channel inactivation kinetics.
Here, we demonstrate that the amino-terminal interaction site of
1A is required for fine tuning the voltage dependence of
activation. The NTA interaction with
4
appears to limit the amplitude of the hyperpolarizing
-induced shift
of channel activation. By this unique mechanism, it can be predicted
that the
3-containing P/Q channel subtype is activated more easily than the
4-containing P/Q channel subtype.
In addition, secondary interaction sites may serve to protect or
uncover phosphorylation sites in the
1A subunit, thereby
altering the regulatory input of these. Another obvious possibility is
that they play a role in the antagonistic relationship between the
subunit and G
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),
G
also interacts with the carboxyl-terminal domain of
1A,
1B, and
1E and that the amino terminus has recently been recognized as another determinant for G
regulation in
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
subunits
also play a role in the surface targeting of
1 and
2
(14), an interesting possibility is that specific
subunits serve to target
1 subunits to specific
regions of the cell surface.

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|
Fig. 8.
Schematic
1A- 4
interactions. Shown are the AID-BID anchoring site (As)
implicating the I-II loop of 1A and the first 30 residues of domain IV of 4; and the two secondary
interactions sites, the first comprising NTA
(Ss1) and the second CTA (Ss2),
involving overlapping regions of domain V of 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.
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,
1 interaction domain;
BID,
interaction domain;
GST, glutathione S-transferase;
PCR, polymerase chain reaction;
MBP, maltose-binding protein;
35S-
4,
[35S]methionine-labeled
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
1A, and
GST-NTA,2-52 for the truncated form of this which contains
only residues 2-52.
 |
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