From 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
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ABSTRACT |
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The voltage-gated calcium channel subunit is
a cytoplasmic protein that stimulates activity of the channel-forming
subunit,
1, in several ways. Complementary binding
sites on
1 and
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
subunit. We describe here the characterization in vitro of
a second interaction, involving the carboxyl-terminal cytoplasmic
domain of
1A and showing specificity for the
4 (and to a lesser extent
2a) isoform. A
deletion and chimera approach showed that the carboxyl-terminal region
of
4, poorly conserved between
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.
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INTRODUCTION |
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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 1 subunit,
the ionic pore of the channel, and two auxiliary subunits,
and
2
, that confer native biophysical and pharmacological properties to the channel. These subunits are encoded by at least six
1, four
, and one
2
gene, for which
numerous splice variants have been identified (3).
The subunit is a cytoplasmic protein of 52-78 kDa that, when
coexpressed with the
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
vary in
importance, depending on the combination of channel subunits studied.
Although
regulation seems to be highly conserved from
1 to
4 and on
1S to
1E, some important differences between these various
isoforms have nevertheless been noted. The different
subunits
produce consistently different channel inactivation behaviors,
3 producing fast inactivation,
2 slow
channel inactivation, and
1 and
4 more
intermediate behaviors (9-11). The
effect also appears to be
1 isoform-dependent; the
-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
1 and
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
1 and
subunits (17, 18).
AID1
(
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 (
subunit interaction
domain), a 30-residue region in the second conserved domain
of the
subunit (domain IV in Fig. 5A). AID and BID
appear to be essential for the subunit interaction and regulation by
subunits (17). Point mutations in AID and BID that disrupt this
primary interaction also totally inhibit channel regulation by
,
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
subunit. The deletion approach used to identify the BID site revealed
that it may not carry all the current stimulatory function of
, 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
subunits over 200 residues and
that, in addition, toward the amino-terminal of
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
-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 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
1 and
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
subunit in the carboxyl-terminal domain of
1A, which is the largest of the cytoplasmic regions and
shows considerable degeneracy of sequence homology between
1 subunit types and splice variants. We demonstrate that
the carboxyl-terminal sequence of
4 specifically
interacts with this region of rabbit
1A (BI-2) and is
required for a proper regulation of channel inactivation.
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EXPERIMENTAL PROCEDURES |
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Preparation of Fusion Proteins--
Regions of the rabbit brain
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
1A (11) were prepared in the same way.
In Vitro Translation of Subunits--
subunit cDNA
clones used were rat
1b (L11453), rabbit heart
2a (X64297), rabbit heart
3 (M88751), and
rat brain
4 (A45982). Truncated derivatives of
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
3 (1-360) and
4 (402-519), a two-step PCR approach was used with the
following primers:
3, forward:
5
-ACGTAAGCTTACCATGGATGACGACTCGTAGGTGCCC-3
, reverse: 5
-GCTTGTGTGGGTGGCGCGCCAGTAAACCTCTAGGTA-3
; and
4,
forward: 5
-GAGGTTTACTGGCGCGCCACCCACACAAGCAGTAGC-3
, reverse:
5
-CGCGGATCCTCAAAGCCTATGTCGGGAGTCATGGCTGCATCC-3
. The reverse
primer for
3 and the forward primer for
4
contain complementary sequences, allowing annealing of the two PCR
products to give the template for the second round of amplification,
using the
3 forward and
4 reverse
primers. Restriction sites HindIII and BamHI were
included in the external primers, allowing subcloning into
pCDNA3.
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 ofElectrophysiological Recordings--
Stage V and VI
Xenopus oocytes were injected with BI-2-specific mRNA
(400 ng/µl) in combination with 4-specific mRNA
(100 ng/µl) or truncated
4
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.
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RESULTS |
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Purified GST fusion proteins carrying amino acids 1889-2126
(GST-1889-2126A) and 2090-2424
(GST-2090-2424A) of the 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
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
1A
(GST-AIDA).
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Analysis of the binding of various concentrations of
GST-2090-2424A to 35S-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
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-
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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We next tested whether the AID-BID interaction had any effect on the
interaction between GST-2090-2424A and 4.
In the presence of a 21-amino acid synthetic peptide containing the AID
sequence of
1A, specific binding of
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
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
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
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
4 for GST-2090-2424A.
To identify the region of the carboxyl-terminal domain that interacts
with 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
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-
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
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
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
interaction to AID, which relies on only three crucial AID residues (14).
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All four 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
1 subunit tested. Functional differences among
subunits may be a reflection of the
differing capacities of
isoforms to form secondary interactions with the
1 subunit concerned. We therefore tested
whether the interaction observed between
4 and
GST-2090-2424A also existed for other
subunits
translated in vitro. Fig. 4
shows a comparison of binding of 35S-
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.
4
interacts with GST-2090-2424A with a high affinity,
showing maximal binding at 1 µM fusion protein
concentration.
2a binds with a much lower affinity,
showing only limited binding at 10 µM fusion protein concentration, while binding of
3 and
1b
is insignificant even at this concentration of fusion protein.
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The differences in interaction affinity observed for different subunits and the fact that the main regions of sequence divergence among
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
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
1A under investigation (Fig. 2B). Deletion of
the amino-terminal 48 amino acids of
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
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
4
N,C. The results show that there was no
difference between
4
C and
4
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
3 was replaced by the corresponding region of
4 (Fig. 5B). The inability of
3 to interact with either
1A fusion
protein was successfully "rescued" by replacement of this region,
resulting in a binding capacity approaching that of the full-length
4 subunit.
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Interestingly, the results obtained with the full-length, truncated,
and chimera 4 were very similar for the two fusion
proteins assayed. This further suggests that the carboxyl terminus of
1A forms a single binding site and not several sites
that would interact independently with diverse regions of
4.
Since deletion of carboxyl-terminal sequences of the 1C
channel induces important modifications in channel gating and opening probability, we determined the functional importance of this
1A-
4 interaction by expression in
Xenopus oocytes. Comparison of the effects of the
full-length
4 and
4
C revealed that the
carboxyl terminus of the
subunit had little influence on the
biophysical properties of the
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
1A
4 and
1A
4
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
1A
4 and
1A
4
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,
1A
4) and
28.1 ± 4.7 mV (n = 6,
1A
4
C). Interestingly, there was a
small but significant change in the rate of inactivation kinetics
produced by the
4 carboxyl-terminal deletion. Cells
injected with
1A
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
4
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
4 may actually contribute to a slowing in inactivation
kinetics upon association to the carboxyl terminus of
1A.
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DISCUSSION |
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We describe here the identification of a new interaction site
between the 1A calcium channel subunit and the
4 subunit. The interaction is of low affinity compared
with that between the AID site on the I-II cytoplasmic loop of
1 subunits and the BID site of
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
subunits would be associated to a single
1 (e.g. as
proposed by Tareilus et al. (20)), seems unlikely for
several reasons. First, the molar ratio between
1 and
subunits is 1:1 in purified channels (2, 23). Second, studies of
the AID-BID interaction revealed that
subunits with affinities
lower than 100 nM for AID do not associate with
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
subunits containing disruptive BID mutations fail to modify channel properties, ruling out additional interactions in these conditions.
The carboxyl-terminal tails of 1 subunits play various
roles in channel function. In the
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
1A
carboxyl-terminal and
4 carboxyl-terminal sequence that
may be of functional relevance. In
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, 4 shows a
much greater affinity for the carboxyl-terminal region of
1A than does
2a, while no interaction is
detected for
1b or
3. These differences
in affinity suggest a functional significance that may help to explain the differences in functional effects seen for different combinations of
1 and
subunits. In light of this, it is
interesting that
4 is coexpressed in the same brain
regions as is
1A, particularly in the cerebellum (21,
27), and is the major
subunit associated with the
1A
subunit in the P/Q channel-type (16). A similar interaction has
recently been reported between an
1E splice variant and
2a (20). It will be interesting to see whether this
interaction also displays a specificity for a particular subset of
subunits.
The advantage of a form of subunit specificity in the
1-
association remains largely to be investigated.
Our data suggest that a secondary interaction site that favors
4 association to BI-2 rather than
3, the
other predominant
in brain (28), could be determinant in underlying
subtle kinetic differences induced by the various
subunits. Besides
obvious functional differences, specific
1-
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
subunits are crucial to cell surface localization of
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 subunit interacting with the carboxyl terminus of an
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 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
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
1A splice variant, BI-1, may not interact with
4. Generally, it remains to be seen whether the splicing
occurring in the carboxyl-terminal tail of the
1 subunit
plays a role in the specificity of the secondary
1-
interaction. Two case scenarios can be discussed. The first is that any
deletion or insertion may modify the regulatory input of the associated
subunit at that location without modifying the type of
subunit
associated. The second possibility is that splicing modifies the
1-
interaction specificity and that it favors the
association of another type of
subunit, presumably to specify a
different membrane targeting of the channel. In the case of
1A, it would be interesting to see whether the carboxyl terminus of BI-1 interacts with
3, the other predominant
subunit known to interact with
1A in the brain (16).
Our data also shed new light on data obtained by other groups that
report a lack of impact of
1 carboxyl-terminal
alternative splicing on
subunit regulation (for instance in
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
1 and
subunits.
It is becoming increasingly obvious that a wide range of neurological
and motor diseases result from mutations in the 1 or
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
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
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
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
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- association on the affinity of the interaction between the
carboxyl termini of
1A and
4 probably
reflects the absence of the remainder of the
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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).
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,
1 subunit interaction domain; BID,
subunit
interaction domain; PCR, polymerase chain reaction; GST, glutathione
S-transferase; PAGE, polyacrylamide gel
electrophoresis.
2 M. De Waard, unpublished data.
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REFERENCES |
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