(Received for publication, September 1, 1995; and in revised form, October 19, 1995)
From the
The subunit of the voltage-dependent Ca
channel is a cytoplasmic protein that interacts directly with an
subunit, thereby modulating the biophysical
properties of the channel. Herein, we demonstrate that the
subunit of the N-type Ca
channel associates
with several different
subunits. Polyclonal antibodies specific
for three different
subunits immunoprecipitated
I-
-conotoxin GVIA binding from solubilized rabbit
brain membranes. Enrichment of the N-type Ca
channels
with an
subunit-specific monoclonal antibody showed
the association of
,
, and
subunits. Protein sequencing of tryptic peptides of
the 57-kDa component of the purified N-type Ca
channel confirmed the presence of the
and
subunits. Each of the
subunits bound to the
subunit interaction domain with similar high
affinity. Thus, our data demonstrate important heterogeneity in the
subunit composition of the N-type Ca
channels,
which may be responsible for some of the diverse kinetic properties
recorded from neurons.
Voltage-dependent Ca channels are essential
for regulating Ca
concentrations in many cells. Based
upon electrophysiological and pharmacological properties, these
channels have been classified into five major groups (L, N, T, R, and
P/Q types)(1, 2) . L-type Ca
channels are central to excitation-contraction coupling in
skeletal and cardiac muscle, while T-type channels are involved in
pacemaker activity. The N-, P/Q-, and R-type Ca
channels are found predominantly in the central and peripheral
nervous systems and have major roles in controlling neurotransmitter
release. The skeletal muscle L-type and the brain N-type Ca
channels have both been purified. Although functionally distinct,
these have similar subunit compositions (
,
, and
), with a variable channel-specific
subunit (
or 95 kDa, respectively)(3, 4) . The
genes encoding the
pore-forming subunits have been
separated into six groups (S, A, B, C, D, and E), each containing
multiple splice variants, while the
subunits have been classified
into four major classes (namely
,
,
, and
), also containing several
splice variants(5) . Recent studies have identified
complementary interaction domains on the
and
subunits(6, 7) . The
subunit regulates channel
activity by binding to a highly conserved portion of the cytoplasmic
linker of the I-II loop of all
subunits, known
as the
subunit interaction domain (AID). (
)The
subunit's interaction site, known as the
subunit interaction domain, encompasses
30 amino acids in
the amino-terminal portion of the second highly conserved domain. In vitro binding studies have shown that an
subunit binds a single
subunit in a 1:1
stoichiometry(8) .
N-type Ca channels are
involved in regulating neurotransmitter release in the central and
peripheral nervous systems and in controlling endocrine secretion.
These also serve as autoantigens in paraneoplastic neurologic disorders
and may be the target of pathogenic autoantibodies responsible for
autonomic dysfunction in the Lambert-Eaton myasthenic
syndrome(9) . Electrophysiological analysis of the N-type
Ca
channels in different neurons has revealed an
unusual degree of diversity in the rates of
inactivation(10, 11, 12) . The structural
basis of this functional diversity is not understood. Herein, we
examine which
subunits are associated with the N-type
Ca
channels from rabbit brain using a monoclonal
antibody specific for the
subunit and polyclonal
antibodies for three different
subunit genes. Several lines of
evidence are presented that demonstrate that there is heterogeneity in
the
subunit of native N-type channels, and this may account for
some of the functional diversity of channel kinetic properties recorded
from neurons.
The N-type Ca channel subunit composition
was investigated using a number of different antibodies. Saturating
amounts of four
subunit-specific antibodies were determined by
incubating increasing quantities of these antibodies with a constant
amount of
I-
-CTx GVIA-labeled receptors to
establish the maximum immunoprecipitation of solubilized rabbit brain
N-type channels (Fig. 1A). The maximum
immunoprecipitation by a variety of antibodies raised against several
components of voltage-dependent Ca
channels was
subsequently compared. Polyclonal antisera against the purified N-type
Ca
channel (Sheep 46) precipitated the
I-
-CTx GVIA receptors and provided the 100% control
value (Fig. 1B). A second polyclonal antibody (Y006)
raised against the II-III loop of the
subunit
precipitated 93 ± 1.8% of the
I-
-CTx GVIA
receptors with respect to the Sheep 46 antibodies. This confirmed the
presence of the
subunit in all N-type Ca
channel oligomers. In contrast, a monoclonal antibody (IIC12)
raised against the purified skeletal muscle dihydropyridine receptor (3) was included as a negative control and was shown to
precipitate negligible amounts (<3%) of
I-
-CTx
GVIA receptors. In addition, a polyclonal antibody specific for the
subunit (Rabbit 140) did not sediment significant
amounts of the N-type channels and served as a second negative control.
Figure 1:
Immunoprecipitation
of I-
-CTx GVIA binding with subunit-specific
antibodies. Aliquots (
20 mg) of rabbit brain membranes were
labeled with
I-
-CTx GVIA in the presence and
absence of 1000-fold unlabeled toxin and solubilized as described under
``Experimental Procedures.'' A, to determine the
maximum amount of antibody required to sediment a constant amount of
solubilized
I-
-CTx GVIA-labeled receptors,
increasing quantities of antibody coupled to protein G Sepharose were
incubated with an aliquot of the labeled channels (1 ml) overnight at 4
°C. The beads were washed three times with 10 mM HEPES/NaOH, pH 7.5, 0.1 M NaCl, and 0.1% (w/v) digitonin
containing five protease inhibitors, and immunoprecipitation was
quantified by
-counting. B, saturating concentrations of
each antibody coupled to protein G-Sepharose were incubated with
solubilized
I-
-CTx GVIA receptor complexes (
1
ml) overnight at 4 °C. The beads were washed as described above and
quantified by
-counting. The amount of precipitation in each case
was determined relative to that of Sheep 46 (Sh46; 100%).
IIC12 is a mAb raised against the skeletal muscle dihydropyridine
receptor.
Saturating amounts of each of the subunit antibodies were used
to precipitate the relative amounts of each
subunit associated
with the
I-
-CTx GVIA receptor. Consistent with
previous observations(14) , a polyclonal antibody specific for
the
subunit precipitated the largest amount of toxin
binding (56.1 ± 8.3%), although a
subunit
antibody was previously shown to sediment a larger fraction of the
receptors. In the earlier study, however, the
subunit
antibodies (affinity-purified from Sheep 46) may have been
cross-reactive with the
subunit, which at that time
had not been cloned. In the present study, the
subunit-specific antibody was raised directly against a
C-terminal fusion protein (Sheep 49) and was shown to be specifically
reactive with the
subunit (30) .
Interestingly, the
subunit-specific antibody also
sedimented a significant proportion of receptors (30.5 ± 2.1%),
suggesting that it is a major component of the purified N-type
Ca
channels. Moreover, coincubation of saturating
amounts of the
and
subunit
antibodies with the labeled receptor precipitated 84 ± 0.7%,
which is approximately equivalent to the sum of precipitation by both
sera incubated separately (56.1 ± 8.3% (
)
+ 30.5 ± 2.1% (
)). This further confirmed
the specificity of these antibodies and demonstrated that both
subunits were not present in the same oligomer since saturating
concentrations of both antibodies incubated together did not
immunoprecipitate less than the sum of each antibody incubated
separately.
Notably, the subunit-specific
antibody also precipitated a significant amount of the labeled
receptors after subtracting the nonspecific precipitation (10.3
± 1.6%), suggesting that it also associates with the
subunit of the N-type Ca
channel
in brain. In contrast, the
subunit antibody did not
precipitate significant amounts of
I-
-CTx GVIA
binding over the nonspecific binding, which was reproducibly <3%,
suggesting either that the
subunit may not be
associated with the brain N-type Ca
channel or that
its expression level in brain is too low to detect(26) . These
immunoprecipitation data represent the first evidence that three
different
subunits associate with the
subunit
in the native N-type Ca
channel.
To investigate
the subunit heterogeneity of N-type channels further,
immunoaffinity-purified Ca
channel subunits (4) were separated by SDS-PAGE, electrophoretically transferred
onto Immobilon PSQ membrane, and visualized by Coomassie Blue staining.
The 57-kDa band was excised and digested with trypsin. This generated
seven peptides, which were resolved by reverse-phase HPLC, followed by
Edman degradation (Table 1). Comparison of the sequences with
those in the database showed that peptides 1-3 had >80% amino
acid identity to the
subunit, which confirmed the
previous observation that this subunit is present in the purified
N-type Ca
channel(4) . Peptides 4 and 7
showed >85% amino acid identity to the more recently cloned
subunit (17) and were absent from the
subunit sequence. The remaining two peptide sequences
are present in the second conserved domain of each of the
subunits and could therefore not be specifically assigned to any of the
subunits. These data confirmed that both the
and
subunits are associated with the N-type
Ca
channels. Since the
subunit has
a molecular mass of
72 kDa, it was resolved from the
and
subunits (molecular masses of 57 kDa) by
SDS-PAGE prior to sequencing, which explains why no specific sequences
for this subunit were detected.
The specificity of the polyclonal
antisera that were raised against the C-terminal fusion proteins of the
(Sheep 49) and
(Rabbit 145)
subunits was tested in immunoblot experiments. COS-7 cells, which do
not contain detectable levels of endogenous Ca
channel subunits, were transiently transfected with constructs
encoding the
and
subunits
separately. The cells were harvested, and equal amounts of the protein
were subjected to SDS-PAGE on a 5-16% gel. The proteins were
electrophoretically transferred onto nitrocellulose and probed with
affinity-purified
and
subunit-specific antibodies. Neither antibody recognized any
proteins in the untransfected cells. The resulting immunoblots
demonstrate the specificity of the
and
subunit-specific antibodies since the
antibodies recognized only the protein in the
-transfected cells and none in those transfected with
. The
antibodies were likewise shown
to be specific for the
subunits expressed in COS-7
cells (Fig. 2).
Figure 2:
Determination of the specificity of the
and
antibodies. The specificity of
the affinity-purified
and
subunit
antibodies was determined by transiently transfecting COS-7 cells as
described under ``Experimental Procedures'' with constructs
encoding the respective
subunit. After harvesting the cells,
aliquots (
150 µg) were subjected to SDS-PAGE on a 5-16%
gel, followed by either Coomassie Blue staining (CB) or
immunostaining with affinity-purified
and
subunit antibodies. Ctl, control untransfected
cells.
The subunit composition of the N-type
Ca channel was further established by the development
of a purification scheme using a heparin-agarose column, as was
previously published(4) , followed by immunoaffinity
chromatography using the
subunit-specific mAb CC18
coupled to Avidchrom hydrazide. This monoclonal antibody was raised
against a fusion protein containing the II-III loop of the
subunit. This bound specifically and with high
affinity to
I-
-CTx GVIA receptors, but did not
immunoprecipitate
I-
-CTx MVIIC receptors (9) or react with the fusion protein containing the
II-III loop of the
subunit (Table 2) in
enzyme-linked immunosorbent assay and immunoblotting experiments, even
though there is a low sequence identity in this region between both
subunits(18, 19) . Furthermore, this
mAb did not immunoprecipitate any significant proportions of
[
H]PN 200-110 binding to skeletal muscle triads
(data not shown), which was not surprising since there is very little
homology between the sequence of the II-III loop of the
subunit and the other
subunit
genes (
,
,
, and
subunits), so the possibility of cross-reactivity of
mAb CC18 in either immunoblotting or immunoprecipitation experiments
with any other
subunit was highly unlikely.
Rabbit
brain membranes were first labeled with I-
-CTx
GVIA, solubilized in high ionic strength buffer containing 1% (w/v)
digitonin and a mixture of five protease inhibitors, and applied to a
heparin-agarose column. Elution of the column with 0.7 M NaCl
resulted in
10-fold enrichment of the channels (Fig. 3A). The peak of channel activity was pooled and
subsequently applied to the mAb CC18-Avidchrom column. Development with
glycine buffer, pH 2.5, yielded a large peak of radioactivity (Fig. 3B) that was pooled and analyzed by SDS-PAGE
followed by Western blot analysis with antibodies to the
subunit and each of the four
subunits. The resulting
immunoblots showed the presence of the broad diffusely stained
subunit, with an apparent molecular mass ranging
from 190 to 230 kDa. This broad band may contain more than one
species since multiple splice variants of the
subunit have been shown to exist(19) .
However, mAb CC18 is raised against the II-III loop and therefore
would be unable to distinguish between the different C-terminal splice
variants. Interestingly, immunoblotting with affinity-purified
,
, and
subunit-specific antibodies (Fig. 3C)
demonstrated the presence of each of these
subunits in the
preparation. As predicted from the immunoprecipitation data, the
subunit was not detected in the enriched preparation
using its specific antibody and the highly sensitive ECL detection
method. Although it cannot be excluded that the
subunit may interact with the
subunit, the
protein levels of this subunit or its level of association with
is too low to detect in brain.
Figure 3:
Analysis of N-type Ca channels immunoaffinity-enriched using mAb CC18 against the
subunit. A, shown is the elution profile of
I-
-CTx GVIA receptors from the heparin-agarose
column. The column was developed with 0.7 M NaCl in buffer A,
collecting 5-ml fractions. B, shown is the elution profile of
the mAb CC18 immunoaffinity column. The eluate from the heparin-agarose
column was loaded onto this antibody column, and enriched channels were
eluted with 50 mM glycine buffer, pH 2.5. C, the mAb
CC18 immunoaffinity column eluate (
1-2 µg) was resolved
on a 3-12% SDS gel and electrophoretically transferred to
nitrocellulose. The subunit composition of the enriched channels was
then examined by immunoblotting with mAb CC18 and each of the indicated
affinity-purified
subunit antibodies. Molecular mass markers (in
kilodaltons) are shown to the left.
Recent
identification of the interaction domains between the and
subunits has allowed the development of an assay for
studying the specific association of these subunits in vitro.
The binding affinity of the AID
fusion protein to in
vitro translated
S-labeled
subunits from each
of the four genes was measured. Interestingly, the AID
fusion protein interacted with the
(K
= 4.7 nM; 90% of total
binding capacity),
(K
=
4.8 nM; 98% of total binding capacity), and
(K
= 7.26 nM; 91% of total
binding capacity) subunits with similar high affinities (Fig. 4). Unlike
,
, and
, the
subunit appeared to bind to
two sites, one with high affinity (8.4 nM; 63% of total
binding capacity) and the other with low affinity (444 nM; 55%
of total binding capacity). The lower binding affinity may have been
due to the binding of some proteolyzed forms of the
subunit that were generated during the synthesis of the probe, as
was previously shown(8) . These data demonstrate that the
AID
fusion protein can interact with each of the
subunits with similar high affinity, unlike the binding to the
AID
site, which showed a 20-fold lower affinity for
than for
(8) .
Figure 4:
Analyses of AID-glutathione S-transferase fusion protein binding to several
subunits. Increasing concentrations of AID
-glutathione S-transferase fusion protein (0.1 nM to 1
mM) were coupled to GSH-Sepharose for 1 h and then incubated
with 0.7-1.3 pM
S-labeled
subunits
for 15 h at 4 °C. The data were fitted using the GraFit fitting
program, yielding apparent K
values of
4.7 nM (
), 4.8 nM (
), 7.6 nM (
), and 8.4
and 444 nM (
). Scatchard plots were fitted by
linear regression (insets). B/F,
bound/free.
Although
several in vitro expression studies using recombinant protein
have shown that subunits form functional
Ca
channels with variable channel kinetics depending
upon which
subunit is
coexpressed(19, 20, 21, 22, 23, 24) ,
this is the first report to demonstrate directly that different
subunits are associated with the
subunit in the
native N-type Ca
channel. The initial suggestion that
only the
subunit was present in the N-type
Ca
channel was made prior to the discovery and
cloning of the
subtype, which we have demonstrated
here to be a significant component of the N-type Ca
channel by immunoprecipitation, internal sequence information,
and Western blotting analysis. Data presented herein revealed that
there is more diversity in the N-type Ca
channel
subunit composition than was originally indicated. In contrast, the
subunit of the skeletal muscle dihydropyridine
receptor appears to associate only with the
subunit
(data not shown) since this is the only
subunit known to be
expressed in skeletal muscle tissue(25) . In neurons, the
levels of expression of each
subunit may, in part, determine the
apparent specificity of association between the
subunit and various
subunits. Our data support this
hypothesis since immunoprecipitation of four different
subunits
with specific antibodies correlates well with the relative amounts of
each
gene expressed in brain, the most abundant being
and
, with smaller quantities of
and negligible proportions of the
subunit(26) . It is thus possible that in different
tissue sources, the levels of expression of certain
subunits
determine the levels of association with the
subunit.
The association of different subunit combinations as a method of
generating subtle differences in channel properties has been reported
for other ion channels. The neuronal voltage-dependent
-dendrotoxin-sensitive K
channels form
hetero-oligomers with various combinations of four
subunits with
and without four ancillary
subunits(27) . In the
ligand-gated
-aminobutyric acid type A receptor, particular
subunits have also been shown to associate with several different
subunits in vivo(28) , thereby creating more channel
heterogeneity. In conclusion, the generation of different N-type
Ca
channel oligomers that differ in their
subunit composition may account for some of the functional diversity of
these channels in the nervous system.