From the
In voltage-dependent Ca
Voltage-dependent calcium channels are a primary pathway for
Ca
Ca
The dissociation of the
The
We thank Dr. T. Tanabe for providing the
channels, the
subunit interacts with the
subunit via a cytoplasmic
site. A biochemical assay has been developed to quantitatively describe
the interaction between both subunits. In vitro synthesized
S-labeled
subunits specifically
bind to a glutathione S-transferase (GST) fusion protein
containing the
interaction domain (AID
,
located between the amino-acids 383 and 400 of the cytoplasmic loop
between the hydrophobic domains I and II). Kinetic analysis
demonstrates that the association of
S-labeled
subunit to the AID
GST fusion protein
occurs with a fast rate constant at 4 °C. The binding is almost
irreversible as demonstrated by the absence of dissociation observed
after an 8-h incubation with an 18-amino acid synthetic AID
peptide. The
-
binding site does not seem
to be a target for cytoplasmic regulation. The interaction is mostly
unaffected by changes in ionic strength, pH, and Ca
concentration or by protein kinase C phosphorylation. The
specificity of subunit interaction in voltage-dependent Ca
channels was also followed by saturation analyses. The data
obtained show that the AID
GST fusion protein binds to a
single site on the
with an apparent
K
of 5 nM. The affinities of
AID
GST fusion protein for various
subunits was
measured and demonstrate that
subunits associate with different
affinities to each
interaction domain. The rank order
of AID
affinity for each
subunit is as follows:
>
>
. The binding of the
subunit to
subunit can be inhibited in vitro by the AID
synthetic peptide with an apparent K
of 285 nM. This interaction can also be prevented in
heterologous Ca
channels by the injection of the
AID
GST fusion protein into Xenopus oocytes.
Our results demonstrate that the site of interaction between AID and
subunit is responsible for anchoring the
subunit to the
subunit and thus allowing the
subunit to modify
Ca
channel activity.
entry into cells, thereby allowing the activation
of numerous cellular processes
(1) . Several classes of
Ca
channels have been distinguished based upon their
functional and pharmacological properties
(2) . N-type and P-type
Ca
channels are found in numerous central and
peripheral neurons and play a key role in the control of
neurotransmitter release (3). L-type Ca
channels are
involved in excitation-contraction coupling in skeletal and cardiac
muscle, whereas T-type Ca
channels are implicated in
pacemaker activity. Two subtypes of these channels have been purified,
the high voltage-activated skeletal muscle L-type channel
(4) and the brain
-conotoxin GVIA-sensitive N-type
channel
(5) . Both Ca
channels are a complex of
three structurally conserved (
,
, and
) and one more variable (
or
95-kDa protein) subunits. The
subunit contains the
pore of the channel and constitutes the receptor of most drugs and
toxins that regulate channel activity. The
subunit is a 160-kDa glycosylated disulfide-linked complex of two
proteins (
and
) that are associated via
disulfide bonds.
subunits are smaller proteins of about
52-76 kDa
(6, 7, 8) . They are entirely
cytoplasmic and, like the
subunit, are substrates for
various protein kinases
(9) . Previous biochemical analyses of
these Ca
channels have demonstrated a very tight
interaction between the
and
subunits. It was
found that both subunits are co-localized in the transverse tubular
membrane of skeletal muscle
(10) ,
co-immunoprecipitate
(7) , and also co-purify from skeletal
muscle
(4) and from brain
(5) .
channel subunits have been separated into distinct classes based
on molecular differences in
(classes S, A, B, C, D,
and E) and
(
,
,
, and
) subunits
(6) .
Additional molecular diversity is introduced by alternative splicing
from these genes
(11, 12, 13) . In most cases,
expression of these
subunits alone is sufficient to
form calcium-selective and voltage-dependent channels. However, the
coexpression of both
and
subunits is
likely required for the membrane targeting and/or the proper
conformation of the
subunit, thereby facilitating its
functional expression
(14, 15) . Both of these ancillary
subunits also affect the channel gating by regulating the
voltage-dependence and kinetics of its activation and
inactivation
(16, 17, 18, 19, 20, 21) .
Interestingly, despite the extreme functional and molecular diversity
of voltage-dependent Ca
channels, it was recently
demonstrated that the structural and functional importance of subunit
interaction sites is well conserved among these channels. Analysis of
the
subunit regulation of voltage-dependent
subunits has led to the identification of a primary interaction
site between both subunits
(20, 21) . The
subunit
binds to a cytoplasmic sequence that is highly conserved and located on
the cytoplasmic linker between repeats I and II of all functionally
distinct
subunits (Fig. 1a). To
facilitate the description of this site, we have called it the
subunit interaction domain or
AID.
(
)
AID represents an 18-amino acid sequence
of which only 9 amino acids are conserved among
subunits (Fig. 1b). Thus, the 9 nonconserved
residues make the AID sequence unique to each
subunit
isoform. In addition, the corresponding interacting domain on the
subunit was identified as a 30-amino acid sequence in the N terminus of
the second highly conserved domain of the
subunit
(21) .
This site is responsible for the anchoring of the
subunit to AID
and is required for current stimulation and hyperpolarizing shift in
activation. We have named this second site the
interaction domain
or BID. BID is even more conserved among
subunits, with a minimum
of 76% amino acid identity than AID is among
subunits. The overall amino acid identity of BID was, in fact,
lowered by the discovery of more variability in the N-terminal sequence
of a housefly
subunit
(22) . Both AID and BID are required
for the binding of the
subunit to the
subunit
and for current regulation. The identification of these two domains in
both
and
subunits therefore provides a unique
opportunity to characterize the biochemical properties of this subunit
interaction. The results reported here establish the basis of a
detailed analysis of the
-
interaction, which is
aimed at resolving the structural determinants involved in the
specificity of subunit recognition in voltage-dependent Ca
channels.
Figure 1:
Diagram of -
interaction site. a, transmembrane folding models and
interaction site of the Ca
channel
and
subunits as determined by primary structure analyses.
Cylinders represent predicted
-helical segments in the
transmembrane regions of the
subunit and in the
peripherally associated
subunit. b, consensus
(AID) and
(BID) interaction
sites. The AID and BID sites describe the region of
-
interaction where X is a capital letter
(S, A, B, C, D, or E) that identifies the genes from which the
subunit originates, N is a number (1, 2, 3,
or 4) that identifies the genes from which
subunit originates,
and y is the splice variant of that gene. The sequences of two
AID proteins used throughout this manuscript are reproduced; the
18-amino acid AID
peptide (amino acids 383-400 of the
subunit) and the 50-amino acid AID
GST
fusion protein (amino acids 369-418). The conserved amino acids
are in filledlettering.
Reagents
TNT coupled reticulocyte lysate
system was purchased from Promega. The peptide corresponding to the
amino acid sequence between 383 and 400 of the rabbit brain
subunit
(23) was synthesized at the
biopolymers facility of the Howard Hughes Medical Institute (University
of Texas Southwestern Medical Center).
Isopropyl-1-thio-
-D-galactopyranoside was from
Life Technologies, Inc.; reduced glutathione was from U. S. Biochemical
Corp. [
S]Methionine was from Amersham Corp., and
glutathione-Sepharose 4B and protein G-Sepharose were from Pharmacia
Biotech Inc. All other chemicals were of reagent grade.
In Vitro Translation of
The
Subunits
S-labeled
subunit probes were synthesized by coupled
in vitro transcription and translation using the TNT system
(Promega). Four cDNA clones were used: rat brain
(GenBank accession number X61394), rabbit heart
(X64297), rabbit heart
(M88751), and rat brain
(L02315). The concentration of the probe in the
lysate after synthesis was determined by trichloroacetic acid protein
precipitation in the presence of 2% casamino acids followed by
scintillation counting. Control experiments demonstrate that between
6.3 and 8.4% of the free radioactive
[
S]methionine is incorporated into newly
synthesized
subunits.
Overlay Experiments
The glutathione
S-transferase (GST) fusion protein epitope of the subunit
(23) was constructed and induced as described
previously
(21) . Crude Escherichia coli DH5
cell
lysate containing the
GST fusion protein was
pelleted and resuspended in equal volume of phosphate-buffered saline
(PBS; 150 mM NaCl, 50 mM sodium phosphate (pH 7.4)).
Samples of the lysate were electrophoretically separated on 3-12%
gradient SDS-polyacrylamide gel and electrotransferred to
nitrocellulose. The blots were blocked for 30 min with 5% nonfat dry
milk in PBS. The probes were overlaid in PBS (1 µl/ml) and
supplemented with 5% bovine serum albumin and 0.5% nonfat dry milk at
different concentrations and times. The transfers were washed 1 h with
5% bovine serum albumin in PBS at room temperature, air-dried, and
exposed to film (Kodak X-Omat AR) for specified times.
Immunoprecipitation of
mAb VD2S-labeled
Subunit
ascites
(7) were
incubated in PBS overnight at 4 °C with protein G-Sepharose beads.
These beads were washed 3 times with PBS and equilibrated in
immunoprecipitation buffer containing 0.3 M NaCl, 20
mM HEPES, 1 mM benzamidine, 0.23 mM
phenylmethylsulfonyl fluoride (pH 7.4). In vitro translated
S-labeled
subunits (1-2 µl/ml) were
incubated with saturating concentrations of VD2
protein
G-Sepharose beads. After a variable amount of incubation time at 4
°C, the beads were centrifuged and washed 4 times with
immunoprecipitation buffer, and the radioactivity bound to the beads
was analyzed by liquid scintillation counting. Nonspecific binding was
determined by measuring the radioactivity bound to control protein
G-Sepharose beads.
Purification of GST Fusion Proteins
Large bacteria
cultures containing the plasmids coding for control GST and GST fusion proteins (wild-type and mutant
sequences containing the amino acid motif that binds
subunits) were inoculated with small overnight cultures, grown at 37
°C for 1 h, and induced for 4 h with 1 mM
isopropyl-1-thio-
-D-galactopyranoside. The cells
were pelleted and resuspended in PBS containing 1% Triton X-100 and
sonicated twice for 15 s each. The sonicated material was centrifuged
(12,000 rpm for 12 min on Beckmann JA-17 rotor). At this stage, the
procedure differed for the control GST fusion protein and the wild-type
or mutant
GST fusion proteins. The supernatant of
the control GST fusion protein was incubated with glutathione-Sepharose
beads. In contrast, because more than 90% of the wild-type or mutant
GST fusion proteins remained in inclusion bodies
(thus were largely insoluble), the initial supernatants were discarded,
and the pellets were resuspended in 10 ml of PBS containing 1%
sarcosyl. After a 30-min incubation at 4 °C, the insoluble material
was removed by centrifugation as stated above. Triton X-100 was added
to the supernatants to a final concentration of 2% and, as with the
control GST fusion protein, the supernatants were then incubated with
glutathione-Sepharose beads for 30 min at 4 °C. The beads that
bound either the control GST, wild-type, or mutant
GST fusion proteins were placed in columns and extensively washed
with PBS. The GST fusion proteins were then eluted with 10 mM
glutathione in 50 mM Tris (pH 8.0). Some batches of purified
GST fusion proteins were also concentrated (Amicon) and dialyzed
(Spectrum, 8000 molecular weight cut-off) against PBS at 4 °C to
remove the glutathione that would compete for the reassociation of the
GST fusion proteins to glutathione-Sepharose beads.
Covalent Coupling of Purified Fusion Proteins to
Sepharose Beads
The purified control GST and GST fusion proteins were covalently coupled to cyanogen bromide
(CNBr)-activated Sepharose beads in 100 mM NaHCO
,
500 mM NaCl (pH 8.3) for 3 h at room temperature and at a
protein concentration of 0.5 mg
ml
. The resins
were extensively washed with PBS, and the remaining active groups were
blocked with 0.2 M glycine (pH 8.0) for 2 h at room
temperature.
Binding of the AID
Control and
GST Fusion Protein to
S-Labeled
Subunits
binding assays were performed in 1 ml of PBS
containing 1-3 µl of in vitro translated
S-labeled
subunit and either 30 µl of control
GST or
GST fusion protein covalently coupled to
CNBr-Sepharose beads or variable concentrations of purified control GST
or
GST fusion proteins noncovalently coupled to 30
µl of Sepharose beads. Reaction time and probe concentrations were
varied independently as defined in the figure legends. After
incubation, the beads were centrifuged and washed 4 times with PBS and
counted by liquid scintillation. Nonspecific counts were determined by
measuring the radioactivity bound to control glutathione-Sepharose
beads. Control experiments demonstrate that these nonspecific counts
are mainly due to the association of free
[
S]methionine (not
S-labeled
subunits) to glutathione-Sepharose beads. Also, the nonspecific binding
of [
S]methionine or
S-labeled
subunits to control GST fusion protein itself (up to 10
µM) was negligible. Thus, changing the concentration of
GST fusion proteins bound to glutathione-Sepharose beads did not affect
the amount of nonspecific counts. Also, because in most assays the
concentration of the radioactive probe was not changed (thus also of
[
S]methionine), the nonspecific counts remained
constant. Determination of the total amount of nonspecific counts
showed that it represented between 10 and 20% of the total binding
(nonspecific counts of [
S]methionine binding to
Sepharose beads + specific binding of
S-labeled
subunits to AID
GST fusion protein). All of the experiments
were performed in triplicate and the mean values are shown with
± S.D.
Scatchard Analysis
0.8-1.2 pM of
in vitro translated S-labeled
subunits
(receptor R
) were incubated for 15 h at 4 °C in PBS
with varying concentrations (50 pM to 2.5 µM) of
GST fusion proteins (ligand L) that were
noncovalently coupled to glutathione-Sepharose beads. Total binding and
nonspecific counts were assessed following the procedure described
under ``Binding of the
S-Labeled
Subunit to the
Subunit.'' Specific binding (complex
LR
) was calculated by subtracting the nonspecific counts
from the total binding. The values were then normalized with respect to
their maximal and minimal counts. The affinity of the fusion protein
for the
subunit was calculated according to the function
[LR
]/[L
] =
-1/K
[LR
]
+
[LR
]
/K
where K
is the dissociation
constant, [L
] is the free ligand concentration at
equilibrium, and [LR
]
= 1 is
the normalized maximum specific binding.
Preparation of Xenopus laevis Oocytes and Injections of
cRNAs and GST Fusion Proteins
Stage V and VI oocytes were
prepared as described previously
(21) and maintained in a
defined nutrient oocyte medium
(24) . cRNAs were transcribed
in vitro using T7 ( subunit) or SP6
polymerase (pSPCBI-2 cDNA). 50 nl of various subunit compositions were
injected into each oocyte at the following concentrations: 0.7
µg
µl
(
subunit) and
0.2 µg
µl
(
subunit).
Some batches of oocytes were also coinjected with control GST or
GST fusion proteins at approximately 0.5
µM concentration in the oocyte. Prior to their injection,
these fusion proteins were purified and dialyzed against PBS to remove
any contaminating glutathione.
Electrophysiological Recordings and Data
Analysis
Two-electrode voltage-clamps were performed using a
Dagan TEV-200 amplifier. Voltage and current electrodes were filled
with 3 M KCl and had resistances between 0.5 and 2 megaohms.
The bath solution was clamped to 0 mV, which served as reference
potential. The recording solution had the following composition: 40
mM Ba(OH), 50 mM NaOH, 2 mM KCl,
1 mM niflumic acid, 0.1 mM EGTA, 5 mM HEPES
(pH 7.4) (adjusted with methanesulfonic acid). Records were filtered at
0.2-0.5 kHz and sampled at 1-2 kHz. Leak and capacitance
currents were subtracted on-line by a P/6 protocol. Voltage pulses were
delivered every 10 s from a holding potential of -90 mV to
various test pulses to determine the peak current. Data were analyzed
using PCLAMP version 5.5 (Axon Instruments). All mean values are shown
with ± S.E.
RESULTS AND DISCUSSION
In Vitro Binding Assay for the
In order to
investigate the -
Interaction
-
interaction in a quantitative
manner, we developed an in vitro binding assay. The
GST fusion protein coupled to CNBr-activated
Sepharose beads (AID
CNBr-Sepharose beads) can be used as a
ligand for in vitro translated
S-labeled
subunit. Fig. 2a demonstrates that
S-labeled
subunit binds specifically to
AID
CNBr-Sepharose beads, whereas no specific binding is
detected with an equivalent amount of control GST fusion protein
coupled to CNBr-activated Sepharose beads (GST CNBr-Sepharose beads).
As a control, we found that no binding occurs when the
subunit is
denatured by boiling the probe before use (data not shown). The
identity of the
S-labeled
subunit
recognized by AID
CNBr-Sepharose beads is confirmed by its
immunoprecipitation by VD2
, a monoclonal antibody directed
against the skeletal
subunit, an alternative splice
variant that shares strong sequence identity with the
subunit
(25) . Finally, the AID
CNBr-Sepharose
beads fail to bind the in vitro translated
S-labeled
subunit. We
confirmed that both the AID and BID sequences were specifically
required for the interaction between AID-Sepharose beads and
subunits by using previously characterized mutants of these
domains
(20, 21) . We found that, compared with the
wild-type AID
GST fusion protein, there is a 15-fold
reduction in the maximum binding of 1 µM of
AID
-Sepharose beads to
S-labeled
, a noninteracting AID
mutant in an
overlay assay
(20) (Fig. 2b). Also 1
µM mutant AID
, which shows a significant
degree of structural and functional interaction, bound 58.4 ±
1.7% (n = 3), only 1.7 times less than the maximum
binding by 1 µM wild-type AID
GST fusion
protein. These values are thus in close agreement with a 9- and
1.3-fold reduction in current amplitude stimulation by
subunit observed upon coexpression of the full-length mutants
Y392S
and E400A
subunits,
respectively
(20) . Also, consistent with the results of the AID
mutations, we found that AID
Sepharose beads binds 95.6
± 16.3% (n = 3) of the
S-labeled
that can be immunoprecipitated by the monoclonal
antibody VD2
. In contrast, AID
Sepharose beads
bound 0 ± 2% (n = 3) of
S-labeled
, a noninteracting BID mutant in overlay and
expression experiments
(21) , and 101.6 ± 6.9% (n = 3) of
S-labeled
, an
interacting BID mutant (Fig. 2c). Overall, these results
demonstrate that, in this in vitro bead assay, the binding of
subunits to AID
-Sepharose beads occurs specifically
via the AID and BID sequences.
Figure 2:
Probing the -
interaction with an in vitro binding assay. a,
autoradiogram of a polyacrylamide gel with 4 µl of lysate
containing 1.3 nMin vitro translated
S-labeled
(
),
S-labeled
bound to control GST
CNBr-Sepharose beads (GST),
S-labeled
bound to AID
CNBr-Sepharose beads
(AID), immunoprecipitation of
S-labeled
by a saturating concentration of VD2
protein G-Sepharose beads (VD2), 4 µl of lysate
containing 0.8 nMin vitro translated
S-labeled
(
) and
S-labeled
bound to AID
CNBr-Sepharose beads (AID). b,
S-labeled
subunit bound to 1
µM AID
and AID
-Sepharose
beads expressed as a percentage of
S-labeled
bound to 1 µM wild-type AID
-Sepharose
beads (overnight reaction times). c, wild-type and mutant
S-labeled
bound to 1 µM
AID
-Sepharose beads expressed as a percentage of wild-type
or mutant
S-labeled
subunit
immunoprecipitated (IP) by VD2
(overnight reaction
times). Probe concentration in b and c was 0.38
pM.
Association and Dissociation Kinetics of the
AID
In
order to determine association kinetics, we measured the amount of
GST Fusion Protein to
Subunit
S-labeled
subunit bound to
AID
-Sepharose beads over time as the binding reaction
approached equilibrium (Fig. 3a). Fitting the data with
a hyperbolic function revealed that the AID
association was
monophasic. The calculated association rate constant is
k
= 0.1
min
µM
for the
binding of
S-labeled
to
AID
-Sepharose beads. At 500 nM AID
GST
fusion protein, the concentration used in this experiment, this rate
constant corresponds to a half-time t of about 20 min. The
kinetics of
S-labeled
subunit binding
to a saturating concentration of VD2
protein G-Sepharose
was also measured (Fig. 3b). With a half-time of 61 min,
the association to the antibody was thus 3-fold slower than to
AID
. In both cases, the binding was stable for the duration
of the experiment once it reached its equilibrium value.
Figure 3:
Time course of AID GST fusion
protein association and dissociation to
[
S]
subunit. a,
binding to AID
-Sepharose beads. 500 nM of
AID
GST fusion protein coupled to 40 µl of Sepharose
beads were incubated at 4 °C for various times with 0.32
pM of in vitro translated
S-labeled
probe in a 1-ml reaction volume. The beads were then
washed 4 times with cold PBS, and the association of the probe was
measured by counting. Nonspecific counts increased linearly with time
by 22%/hour (not shown). The highest nonspecific was 15.7 ± 1.4%
(n = 3) of maximum total binding. For each time point,
the specific binding was normalized to the maximum specific binding.
The data were fitted with a hyperbolic function f =
a
t/(t + t) where t is the association time, a = 1.109 (the asymptotic
maximum), and t = 20 min (the time of
half-association). b, binding to VD2
monoclonal
antibody coupled to protein G-Sepharose beads. 40 µl of beads were
incubated at 4 °C for various times with 0.34 pM
S-labeled
in 1 ml of PBS. Nonspecific
counts were 6.8 ± 2.2% (n = 3) of maximum total
binding. The asymptotic maximum is a = 1.03, and the
half-association time is t = 63 min. c, the
association was allowed to reach equilibrium by overnight incubation of
the reactants under similar concentrations than those described in
a. The dissociation was then measured at 4 °C in the
presence of 500 µM AID
peptide. Nonspecific
counts remained fairly constant with a linear decrease of 4%/hour (not
shown). The highest nonspecific binding was 16.1 ± 1.9% (n = 3) of maximum total binding. A semilogarithmic plot was
used to represent the data and the linear regression was fit to the
data.
With
respect to the functional importance of subunits in the
regulation of current properties, we further analyzed the effects of
different agents that may affect the conformation of the
subunit and its ability to associate with AID
CNBr-Sepharose beads. We found that the maximum association of
S-labeled
was not affected by changes
in Ca
concentration (1 nM to 1 mM),
ionic strength (0 to 2 M NaCl), or phosphorylation by protein
kinase C (data not shown). Large variations in pH (4 to 10) could
decrease the maximal association by 42%. This interaction was even
totally abolished at pH 12. However, small variations of pH in
physiological range (6.9-7.5) were without effects. These results
are therefore consistent with previous observations that the purified
N-type Ca
channel complex remains intact at pH
10
(5, 26) . Overall, these data demonstrate that the
AID-BID interaction site is an unlikely target for cell inhibitory
regulation of the channel activity such as those implicated in
Ca
dependent inactivation.
S-labeled
-AID
-Sepharose
bead complex was monitored after the binding reactions reached
equilibrium (with 500 nM AID
GST fusion protein).
The beads were washed twice with PBS to eliminate the free
[
S]methionine and the residual unbound probe.
They were then resuspended in 1 ml of PBS, and the dissociation was
triggered by the addition of an excess (500 µM) of the
competing AID
peptide. This concentration of the peptide is
sufficient to fully inhibit the association of
S-labeled
to AID
CNBr-Sepharose beads (see
Fig. 7
). After various times, the beads were washed twice again
to remove the probe dissociated from AID
-Sepharose beads,
and the amount of
S-labeled
that
remained bound was measured. At 4 °C, the interaction between
AID
and
was almost irreversible, as no
measurable decrease in the amount of
subunit bound to the beads
was observed up to 8 h (Fig. 3c). A similar result was
obtained with AID
-CNBr-Sepharose beads in the absence of
AID
peptide (data not shown). Also, this result is
consistent with the observation that injection of the AID
peptide or AID
GST fusion protein did not induce
dissociation of preformed voltage-dependent Ca
channels expressed in Xenopus oocytes (data not shown).
Figure 7:
AID peptide inhibition of
AID
GST fusion protein binding to
[
S]labeled
subunit. The
probe (0.32 pM) was preincubated for 6 h
with various concentrations of the AID
peptide and then
incubated with AID
CNBr-Sepharose beads. Nonspecific counts
were 18 ± 2.2% (n = 3) of total maximum binding.
The data were fitted with a four-parameter logistic function
f([AID]) = (a - c)/(1
+ ([AID]/K)) + c where a = 1.013 and c = -0.03 are the
asymptotic maximum and minimum respectively, b = 0.75
is the slope parameter, K = 285.7 nM is the
inhibition constant at the inflection point, and [AID] is the
concentration of AID
peptide.
Four Different
Subunits Can Interact with the Same
AID Sequence
S-Labeled in vitro translated
subunits (
,
,
, and
) were incubated overnight with
AID
GST fusion protein from E. coli lysates
separated on SDS-polyacrylamide gels and transferred to nitrocellulose
(Fig. 4). The data demonstrate that all four
subunits were
capable of interacting with the same AID sequence. Thus these overlay
experiments prove that sequence variability among and beyond the BID
sequences of these
subunits does not prevent their interaction
with a single AID sequence. These results confirm recent expression
experiments demonstrating the functional interaction of several
subunit types with the full-length
or
subunits by amplitude stimulation and changes in the
voltage-dependence and kinetics of the current
(21, 27) .
They are also comparable with the results showing that a single BID
sequence can interact with multiple AID sequences (see
Fig. 6b). Altogether, these observations suggest the
possible existence of a cross-reactivity between various
and
subunit isoforms. If these interactions are confirmed
in situ, such a cross-reactivity would have important
consequences on the functional diversity of voltage-dependent
Ca
channels. However, preliminary results based on
the characterization of the subunits that compose the skeletal muscle
L-type and the neuronal N-type Ca
channels have
suggested the existence of a rather strong specificity in the
interaction between
and
subtypes. In these
Ca
channels, the
subunit
(28) interacts specifically with the
subunit
(29) , whereas the
subunit
interacts with the
subunit
(5) . In an attempt
to resolve some of these experimental contradictions and in order to
get a better understanding of the possible molecular basis for the
specificity of
-
interaction seen in purified
Ca
channels, we have analyzed the affinity of various
subunits for the AID
site.
Figure 4:
Four
different subunit gene products interact with AID
expressed as a GST fusion protein. a, autoradiogram of
SDS-polyacrylamide gel of in vitro translated
[
S]methionine-labeled
,
,
, and
subunits.
5 µl of each translation reaction were run per lane. The
film was exposed for 2 h. b, autoradiogram of
S-labeled
subunit overlays on AID
GST
fusion protein immobilized on nitrocellulose. The probe concentrations
were between 0.3 and 0.6 pM. The overlays were exposed for 3
h. Molecular weight standards (
10
) are
indicated in the middle.
Figure 6:
Peptide inhibition of
-
association. a, crude E. coli lysates (70 µl) of control GST, and AID
,
AID
, AID
, and AID
GST fusion
proteins were analyzed on Coomassie Blue-stained 3-12%
SDS-polyacrylamide gel. The sequence of these fusion proteins were
described elsewhere (20). b, autoradiograms of the
corresponding overlays of these fusion proteins with 0.5 pM
S-labeled
subunit probe. The probe was
preincubated for 4 h with 100 µM of a control 12-amino
acid peptide (CCPNVPSRPQAM) of the C-terminal dystrophin related
protein (33) (left) or 100 µM 18-amino acid
AID
peptide (right). The overlays were exposed for
2 h. The positions of the molecular weight standards
(
10
) are shown on the left of the
Coomassie Blue-stained gel and between the two
overlays.
Ligand Binding Properties
Analysis of the binding
of AID to
S-labeled
subunit
demonstrates that the specific binding is saturable and occurs on a
single binding site (Fig. 5). We calculated an apparent
K
of 5.8 nM for the binding of
AID
GST fusion protein to
subunit. It
is, however, likely that the real affinity between
and
subunits is in fact higher than reported here,
considering the use of a fusion protein as the ligand instead of the
full-length
subunit. Scatchard analysis of the
binding of AID
GST fusion protein to other
subunits
confirms the observations of the overlay experiment of
Fig. 4
demonstrating that AID
is able to interact with
all four classes of
subunits. However, this analysis provided
more detailed information than the overlay data. Despite the strong
sequence homology in the respective BID sequence of these
subunits (87% amino acid identity), the binding of AID
GST
fusion protein to all four
subunits occurs with different
apparent affinities. For instance, the binding of AID
GST
fusion protein to the
subunit occurs with a
K
of about 55 nM, which is at
least 1 order of magnitude lower than the high affinity state observed
for
,
, and
subunits. This result strongly points to the importance of the
conformation of
subunits in determining the affinity of the
-
interaction. Also, two binding sites can be
seen with
and
subunits. The
presence of the lower affinity site cannot be due to proteolytic
fragments of the fusion protein since the same purified material was
used for all four Scatchard experiments described in Fig. 5. The
low and high affinity sites seen with
and
subunits were therefore inherent to the
subunit
themselves. Expression experiments with truncated forms of the
subunits have suggested that sequence deletion may induce a lower
affinity of the
subunit for the
channel
expressed in Xenopus oocytes
(21) . Therefore, it is
likely that the binding of partially proteolyzed forms of
subunits accounts for the component of lowest affinity. Consistent with
this interpretation are the observations that (i)
and
were the most sensitive to proteolysis
(Fig. 4a), (ii) synthetic peptides containing the
BID
sequence (i.e.
213-245) could not bind the AID
GST fusion
protein (data not shown), and (iii) very proteolyzed forms of the
subunit could not interact with AID
GST fusion protein
beads (Fig. 2a). Alternatively, we cannot rule out that
the second site of lowest affinity resulted from misfolding of a
fraction of the in vitro synthesized
subunits. Finally,
the Scatchard analyses provide a rank order of binding affinity of each
subunit to AID
GST fusion protein that is
>
>
when taking into account the component of highest affinity. With
the exception of
, this is also the order in which
subunits can be classified for their potency of
current stimulation in oocytes, suggesting that the affinity
between
and
, and the
current stimulation
efficiencies may be correlated (21). Incidentally, these results also
confirm several predictions suggesting that the
subunit is probably associated with a class A
subunit in native Ca
channels expressed in the
cerebellum
(30, 31) . The development of this
quantitative assay capable of analyzing the interaction of various BID
sequences with a given AID sequence represents therefore a unique way
to test the affinity of several
subunits for a given
subunit. This assay combined with an extensive mutagenesis of the
AID and BID sequences, will ultimately prove helpful to the
understanding of the molecular determinants implicated into the
specificity of subunit interaction of native voltage-dependent
Ca
channels
(4, 5) .
Figure 5:
Scatchard analysis of AID GST
fusion protein binding to various
subunits. Various
concentrations of AID
GST fusion protein (50 pM to
1 µM) were precoupled for 1 h to glutathione-Sepharose
beads and then incubated for 15 h at 4 °C with in vitro translated
S-labeled
subunits (0.7 to 1.2
pM) as described under ``Experimental Procedures.''
A saturation curve is shown for
and Scatchard
representation for all
subunits (see inset for
). Linear regression fitting of the data yielded
apparent K values of 5.8 nM (
), 3.5
and 36 nM (
), 55.1 nM
(
), and 2.6 and 76 nM
(
).
Competition of the
Competition experiments were performed with the
18-amino acid synthetic AID-AID
GST Fusion
Protein Interaction by a Synthetic AID
Peptide
peptide. Four hours of
preincubation of the
S-labeled
probe
with 100 µM AID peptide was sufficient to entirely prevent
the subsequent association of
S-labeled
with AID
, AID
, AID
, and
AID
GST fusion proteins immobilized on nitrocellulose
(Fig. 6). Similar results were obtained by using 1
µM AID
GST fusion protein instead of 100
µM AID
peptide (data not shown). In contrast,
the same concentration of a control unrelated peptide had no effect.
These experiments demonstrate that (i) the AID sequence is by itself
able to bind to
subunits without requiring N and C termini
flanking sequences and (ii) the binding of the peptide is also
irreversible. This 18-amino acid sequence is therefore sufficient by
itself to prevent the association between
and
subunits. The AID
GST fusion protein differs from the
AID
peptide by the presence of additional 14- and 18-amino
acid sequences at the N and C termini of the
sequence. In order to understand the role of these nonbinding
sequences in the AID-
interaction, we compared the relative
affinities of the AID
peptide and AID
GST
fusion protein to bind to the
S-labeled
subunit.
S-labeled
probe
was incubated for 6 h with increasing concentrations of AID
peptide (0.5 nM to 100 µM). The mixture was
then incubated overnight with AID
CNBr-Sepharose beads, and
the amount of
S-labeled
able to bind to
the AID
CNBr-Sepharose beads was measured by scintillation
counting. The results demonstrate that the binding of the AID
peptide to
S-labeled
prevents the
association of the probe to the AID
CNBr-Sepharose beads
with a K
of 285 nM
(Fig. 7). Complete inhibition of
S-labeled
association to AID
CNBr-Sepharose beads
by AID
peptide occurs at concentrations above 10
µM. These data show that the association of AID
peptide to the
subunit occurs with a 57-fold lower affinity
than the association of the AID
GST fusion protein to the
subunit. The results suggest therefore that the interaction of
the AID sequence with the
subunit is greatly facilitated by
flanking noninteracting sequences. This probably occurs by the
determination of a more favorable conformation of the AID sequence.
Because of the ability of the AID sequence to prevent the interaction
between the
subunit and the AID GST fusion protein, we further
tried to determine whether the synthetic AID
peptide or the
AID
GST fusion protein were also capable of inhibiting the
native interaction between the
subunit and the full-length
subunit in cells.
Injection of AID
Both AID and BID are essential structural
elements to the functional regulation of voltage-dependent
CaGST Fusion Protein into
Xenopus Oocytes Prevents the Native Interaction between
and
Subunits
entry by
subunits. We have previously
demonstrated that the interaction of these two domains is required for
(i) the subunit assembly of the Ca
channel and (ii)
the biophysical and pharmacological changes by
subunits
(21) . In the case of the
subunit,
the biophysical regulation by various
subunits include a dramatic
stimulation in current amplitude, hyperpolarizing displacements of the
voltage-dependence of activation, and inactivation and modulations in
the kinetics of inactivation
(32) . Injection of 0.5
µM AID
GST fusion protein into Xenopus oocytes sustainably decreased the amplitude of the current carried
by the
subunit (Fig. 8a). There was
an average 9.2 ± 2.7-fold (n = 3) decrease in
current amplitude stimulation by the
subunit that
lasted for at least 5 days after injection of the cRNAs encoding the
and the
subunits
(Fig. 8b). In comparison, the control GST fusion protein
only reduced the
current stimulation by a nonsignificant factor
of 1.09 ± 0.36-fold (n = 3) over the same time
period. Also, the AID
GST fusion protein induces a
depolarizing shift in the current-voltage relationship of activation
(Fig. 8c). The amplitude of the shift equaled 11 mV at
the peak of the current amplitude. Finally, the AID
GST
fusion protein induced a depolarizing shift in the voltage dependence
of inactivation (data not shown). None of these depolarizing shifts
were detected with the injection of an identical concentration of
control GST fusion protein, demonstrating the implication of the
AID
sequence. All of these effects (decrease in current
amplitude and depolarizing shifts in activation and inactivation) go
into directions that are opposite to the effects of
subunits on
the
current
(21, 32) . These results
demonstrate, therefore that the AID
GST fusion protein is
able to durably prevent the association between full-length
and
subunits in native
conditions. In contrast, this association was not prevented by the
AID
peptide, probably because of its lower affinity for the
subunit or because of a reduced stability in oocytes (data not
shown). The Scatchard analyses of the interaction between AID
and various
subunits suggest that probably all AID
sequences are likely able to inhibit the regulatory function of
subunits by diverting their binding activity. However, because of
differences in AID-BID affinities, it is also likely that the AID GST
fusion protein inhibition of the
-
coupling can
be greatly facilitated by the use of the appropriate AID sequence.
Figure 8:
Injection of AID fusion
protein blocks the interaction between full-length
and
subunits in oocytes. a, Ba
current
traces obtained 2 days after expression of
and
subunits without (control) or with (GST or AID) coinjection of 0.5 µM control GST or
AID
GST fusion protein. The holding potential is -90
mV, and the test potential is 10 mV (control and GST)
or 20 mV (AID) to induce the maximum inward current.
b, average Ba
current stimulation induced by
subunit in control oocytes and AID
- or
GST-injected oocytes as a function of expression time. Data are the
average of 64 oocytes. c, average normalized current-voltage
relation for
+ control GST
(filled circle) or
+
AID
(empty circle). The current-voltage
relationship for
is given as a
dashedline. Data were fitted with a modified
Boltzmann function with I
=
(g(TP - E))/(1 +
exp(-(TP - V)/k)) where g = 79.1 microsiemens (control GST) or 17.9 microsiemens
(AID
) is the conductance, E =65
(GST) or 58 mV (AID
) is the reversal potential, TP is the test potential, and k = 4.3 (GST) or 5.2 mV
(AID
) is the range of potential for an e-fold
change around V = -7.6 (GST) or 4.1 mV
(AID
) oocytes. The data and the curve resulting from the
fit of the data were normalized with respect to the maximum current
amplitude reached in each experimental
condition.
In conclusion, expression in various cell types of proteins
containing the AID sequence should prove useful to investigate the
functional importance of subunits in several important
Ca
channel functions such as excitation-contraction
coupling or excitation-secretion coupling. Disruption of the anchoring
of
subunits to
subunits in native
Ca
channels may contribute to the identification of
the molecular targets of various channel regulators, such as G proteins
or endogenous kinases. Finally, the in vitro method described
here for the study of the
-
interaction will also
be useful to the screening of drugs capable of modulating the
anchoring/regulation of
subunits to/of Ca
channels.
subunit interaction
domain; BID,
subunit interaction domain; GST, glutathione
S-transferase; PBS, phosphate-buffered saline.
cDNA, Drs. V. Flockerzi and F. Hofmann for the
cDNA, and Drs. C. Wei and L. Birnbaumer for the
cDNA . We also thank Drs. F. Duclos, D. Jung, and V. Scott for
critical reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.