(Received for publication, April 3, 1995; and in revised form, June 9, 1995)
From the
Biochemical properties of the subunits of
class A brain calcium channels (
) were examined in
adult rat brain membrane fractions using a site-directed anti-peptide
antibody (anti-CNA3) specific for
. Anti-CNA3
specifically immunoprecipitated high affinity receptor sites for
-conotoxin MVIIC (K
100
pM), but not receptor sites for the dihydropyridine isradipine
or for
-conotoxin GVIA. In immunoblotting and immunoprecipitation
experiments, anti-CNA3 recognized at least two distinct immunoreactive
polypeptides, a major form with an apparent
molecular mass of 190 kDa and a minor, full-length form with an
apparent molecular mass of 220 kDa. The 220- and 190-kDa
polypeptides were also specifically recognized by both anti-BI-Nt
and anti-BI-1-Ct antibodies, which are directed against the
NH
- and COOH-terminal ends of
predicted
from cDNA sequence, respectively. These data indicate that the
predicted NH
and COOH termini are present in both size
forms and therefore that these isoforms of
are
created by alternative RNA splicing rather than post-translational
proteolytic processing of the NH
or COOH termini. The
220-kDa form was phosphorylated preferentially by cAMP-dependent
protein kinase, whereas protein kinase C and cGMP-dependent protein
kinase preferentially phosphorylated the 190-kDa form. Our results
identify at least two distinct
subunits with
different molecular mass, demonstrate that they may result from
alternative mRNA splicing, and suggest that they may be differentially
regulated by protein phosphorylation.
In the nervous system, voltage-gated calcium channels are
involved in initiation of activity-dependent events such as
neurotransmitter release, regulation of action potential duration and
frequency, protein phosphorylation, and gene expression (Llinas, 1988;
Tsien et al., 1988; Olivera et al., 1994). Based on
the pharmacological and physiological properties, at least five
distinct types of voltage-gated calcium channels, designated L, N, P,
Q, and T, have been identified (Bean, 1989; Llinas et al.,
1989; Zhang et al., 1993). Voltage-gated calcium channels are
a complex of five subunits: ,
,
,
, and
(Takahashi et al., 1987; Catterall et al., 1988; Campbell et al., 1988).
subunits can function alone as voltage-gated calcium channels
when expressed in Xenopus oocytes or mammalian cells
(Perez-Reyes et al., 1989; Mikami et al., 1989),
whereas coexpression of the other subunits can alter functional
properties of
subunits (Lacerda et al.,
1991; Singer et al., 1991; Varadi et al., 1991; Wei et al., 1991) (reviewed by Isom et al. (1994)). cDNAs
encoding five distinct
subunits of brain calcium
channels have been identified and designated A, B, C, D, and E
(
-
) (
)(Snutch et al., 1990; Snutch and Reiner, 1992; Zhang et al.,
1993; Catterall, 1994a). The class C and class D genes encode L-type
calcium channel
subunits (
and
), which have a high affinity for dihydropyridines
and conduct long lasting Ba
currents. In contrast,
the class A, B, and E genes encode
subunits
(
,
, and
) of
non-L-type calcium channels, which are more distantly related to L-type
calcium channels (23-35% amino acid identity) (Mori et
al., 1991; Starr et al., 1991; Dubel et al.,
1992; Williams et al., 1992a; Fujita et al., 1993;
Niidome et al., 1993; Soong et al., 1993).
forms an N-type calcium channel, which is
neuro-specific and distinguished by high sensitivity to the cone snail
toxin
-conotoxin GVIA (Dubel et al., 1992; Williams et al., 1992a; Fujita et al., 1993).
forms a novel, rapidly inactivating calcium channel, which has
some characteristics of a low voltage activated calcium channel (Soong et al., 1993; Williams et al., 1994).
The class A
calcium channel (also designated BI) was the first non L-type calcium
channel to be cloned, sequenced, and expressed (Starr et al.,
1991; Mori et al., 1991). forms high
voltage activated calcium channels and Northern blot analysis shows
high expression in the cerebellum (Starr et al., 1991; Mori et al., 1991; Sather et al., 1993; Stea et
al., 1994a).
currents expressed in Xenopus oocytes are insensitive to dihydropyridines and
-conotoxin
GVIA, and therefore
subunits may form P-type and/or
Q-type channels (Mori et al., 1991; Sather et al.,
1993; Stea et al., 1994a).
channels
expressed in Xenopus oocytes inactivate slowly or rapidly
depending on the
subunit expressed with them, and are blocked by
-agatoxin IVA purified from Agelenopsis aperta venom at
high concentration and by
-conotoxin MVIIC from Conus magus (Hillyard et al., 1992; Sather et al., 1993;
Stea et al., 1994a). In contrast, native P-type calcium
channels are blocked by low concentrations of
-agatoxin IVA and
by higher concentrations of
-conotoxin MVIIC (Mintz et
al., 1992a, 1992b, Hillyard et al., 1992). The
pharmacological properties of
calcium channels in Xenopus oocytes are distinct from P-type channels, but more
closely resemble those of calcium channels in cerebellar granule cells,
which have been designated Q-type (Randall et al., 1995; Zhang et al., 1993). In the experiments described in this paper, we
used site-directed anti-peptide antibodies against unique sequences in
rat brain
to identify the corresponding polypeptides
and demonstrated that there are multiple isoforms of
subunits that may result from alternative RNA splicing and are
differentially phosphorylated by second messenger-activated protein
kinases.
The purified peptides were coupled through amino groups
with glutaraldehyde to bovine serum albumin (BSA), dialyzed against
phosphate-buffered saline (10 mM NaHPO
(pH 7.4), 150 mM NaCl) and emulsified in an equal volume
of Freund's complete (initial injection) or incomplete adjuvant.
The coupled peptides were injected into multiple subcutaneous sites on
New Zealand White rabbits at 3-week intervals. Antisera were collected
after the second injection and tested by enzyme-linked immunosorbent
assay using microtiter plates with wells coated with 0.5 µg of
peptide (Posnett et al., 1988). Antibodies were purified by
affinity chromatography on the corresponding peptides coupled to
CNBr-activated Sepharose. Two ml of the antiserum were bound to the
column at 4 °C overnight and washed with TBS (10 mM Tris-HCl (pH 7.4), 150 mM NaCl). The bound IgG was eluted
with 3.0 M MgCl
. The affinity-purified antibodies
were then dialyzed against TBS using a Centriprep 30 (Amicon).
Anti-BI-Nt and anti-BI-1-Ct antibodies were generous gifts from Dr.
Masami Takahashi (Mitsubishi-Kasei Life Sciences Institute, Tokyo,
Japan), and these antibodies were produced against peptides
MARFGDEMPARYGGGGAGAA(C) (Leveque et al., 1994) and
(C)RDQRWSRSPSEGREHTTHRQ, which correspond to residues 1-23 and
2254-2273 of the BI-1 cDNA clone encoding a rabbit brain
subunit, respectively (Mori et al., 1991).
The cysteine residue in each peptide was added to facilitate
cross-linking and radiolabeling and is not part of the
subunit sequence.
Determination of I-
-conotoxin GVIA binding was done by incubation of
100 µl of S3 fraction containing 0.2% BSA with 0.06 µCi of
I-
-conotoxin GVIA (2200 Ci/mmol) at a concentration
of 0.27 nM for 30 min on ice. Samples were immunoprecipitated
with 15 µg of affinity-purified anti-CNA3, anti-CNB2, or control
rabbit IgG, and washed four times with TBS, 0.1% digitonin. The matrix
was transferred to vials for
counting. Total
I-
-conotoxin GVIA binding was determined using 100
µl of the labeled S3 fraction in the filter-binding assay described
above for [
H]PN200-110.
I-
-Conotoxin MVIIC binding was determined by
incubation of 400 µl of samples containing 140 µl of WGA
extract, 10 mM Tris-HCl (pH 7.4), and 0.2% BSA with 0.1
µCi of labeled toxin (1300 Ci/mmol) at a concentration of 0.15
nM on ice for 30 min. This was added to 15 µg of
affinity-purified anti-CNA3, anti-CNC1, or control rabbit IgG, coupled
to 2 mg of PAS, and incubated for 4 h on ice in a tilting mixer.
Samples were washed quickly three times in 10 mM Tris-HCl (pH
7.4), 75 mM NaCl, 0.1% digitonin, 0.2% BSA.
I-
-Conotoxin MVIIC binding in the pellet was
counted in a
counter. For the competitive displacement studies,
the unlabeled ligands
-conotoxin MVIIC or
-agatoxin IVA at
concentrations ranging from 10
to 10
M were added to samples with
I-
-conotoxin MVIIC and incubated with
affinity-purified anti-CNA3, and the bound
I-
-conotoxin MVIIC was measured as described above.
For peptide block, 20 µM test peptide was added to the
affinity-purified antibodies and incubated overnight on ice prior to
incubation with samples containing WGA extract.
Biotinylated samples were preabsorbed for 1 h on ice with 300 µl of Sepharose CL-4B and for 2 h on ice with 10 mg of PAS, which was preincubated with 200 µg of control rabbit IgG and washed three times with TBS, 0.1% digitonin in order to remove the nonspecifically binding proteins in the sample. After centrifugation, supernatants were incubated for another 2 h on ice with 10 mg of PAS to adsorb the free IgG dissociated from PAS-control rabbit IgG complex. After centrifugation for 1 min on a table-top centrifuge, the supernatants were collected and incubated with anti-CNA3 (40 µg), anti-BI-Nt (30 µg), anti-BI-1-Ct (30 µg), anti-CNA1 (80 µg), or control antibody (80 µg) for 1.5 h on ice. The immunoprecipitation was performed as described in the section above, and the pellets were extracted for 30 min at 50-60 °C with 20 µl of 1.5% SDS, 50 mM Tris-HCl (pH 7.4), 5 mM dithiothreitol, 1 µM pepstatin A, 2 µg/ml leupeptin, and 4 µg/ml aprotinin, and diluted with 250 µl of Triton buffer (1% Triton X-100, 0.5% BSA, 75 mM NaCl, 25 mM Tris-HCl (pH 7.4), 20 mM EDTA). The supernatant was collected and incubated for 1.5 h on ice with the secondary antibodies anti-CNA3 (40 µg) or anti-CP(1382-1400) (20 µg). Three mg of PAS, pretreated as described above, were added, and the samples were incubated on a tilting mixer for 2.5 h on ice. The immunoprecipitated complexes were pelleted by centrifugation, washed three times with Triton buffer and once in 10 mM Tris-HCl (pH 7.4), and extracted for 30 min at 50-60 °C with SDS sample buffer. After a short centrifugation, the supernatants were loaded onto an SDS-PAGE gel. The proteins were blotted, blocked as described above, and nitrocellulose sheets were rinsed with TBS, 5% BSA, 0.2% Nonidet P-40, and 0.05% Tween 20, and incubated for 1 h at room temperature with streptavidin-biotinylated horseradish peroxidase complex, diluted 1:8000 in TBS containing 0.2% Nonidet P-40 and 0.05% Tween 20. After a 3-h wash with 0.2% Nonidet P-40, 0.05% Tween 20 in TBS (8-9 changes), the blots were developed with the ECL reagent.
[H]PN200-110 is a dihydropyridine that
specifically binds to L-type calcium channels containing
and
(Hell et al., 1993a). At a
concentration of [
H]PN200-110 chosen to saturate
all binding sites in a rat brain homogenate, affinity-purified
anti-CNC1, an
-specific site-directed anti-peptide
antibody, immunoprecipitated 40% of the total
[
H]PN200-110 receptors, whereas anti-CNA3
immunoprecipitated less than 1% of the total
[
H]PN200-110 receptors, a value similar to that
obtained with control rabbit IgG (Fig. 1A). The binding
of
I-
-conotoxin GVIA, a selective blocker of the
N-type calcium channels containing
, was tested
similarly. Affinity-purified anti-CNB2, an
-specific
site-directed anti-peptide antibody, immunoprecipitated over 80% of the
total binding sites in S3 fractions. Under the same conditions,
anti-CNA3 and control rabbit IgG recognized less than 4% of the total
I-
-conotoxin GVIA receptors (Fig. 1B).
-Conotoxin MVIIC blocks Q-type calcium
channels containing
(Sather et al., 1993;
Stea et al., 1994a; Hillyard et al., 1992). A
saturating concentration of
I-
-conotoxin MVIIC was
added to the solubilized and partially purified calcium channel
preparation. Affinity-purified anti-CNA3 antibodies effectively
immunoprecipitated
I-
-conotoxin MVIIC receptors (Fig. 1C). Anti-CNA3 specifically recognized the
-conotoxin MVIIC receptors, since preincubation of 20 µM CNA3 peptide largely blocked immunoprecipitation of them, whereas
the CNC1 peptide at the same concentration did not affect the
immunoprecipitation of
I-
-conotoxin MVIIC receptors
with anti-CNA3. In contrast, anti-CNC1 and control rabbit IgG
immunoprecipitated only small amounts of
I-
-conotoxin MVIIC.
-Conotoxin MVIIC has a
significant affinity for
-conotoxin GVIA binding sites on
(Hillyard et al., 1992). However, anti-CNB2
did not immunoprecipitate detectable
I-
-conotoxin
MVIIC-labeled
when 0.15 nM
I-
-conotoxin MVIIC was used in this
experiment.
Figure 1:
Immunoprecipitation of brain calcium
channels labeled with [H]PN200-110,
I-
-conotoxin GVIA, or
I-
-conotoxin MVIIC. A, rat brain membrane
fraction (S1) was incubated with [
H]PN200-110 at
the concentration of 2.9 nM, solubilized, immunoprecipitated
with anti-CNC1, anti-CNA3, or rabbit control antibodies, and the bound
[
H]PN200-110 was counted (see ``Experimental
Procedures''). Anti-CNC1 was raised against a highly variable site
of the class C L-type calcium channel
subunit (Hell et al., 1993a). Total [
H]PN200-110
receptor sites were estimated by filter binding assay. B, S3
fractions were incubated with
I-
-conotoxin GVIA
(0.27 nM) and immunoprecipitated with anti-CNB2, anti-CNA3, or
control IgG. Anti-CNB2 is specific for class B N-type calcium channel
subunit (Hell et al., 1994). C,
calcium channels were purified from the S3 fraction by chromatography
on WGA-Sepharose, incubated with
I-
-conotoxin MVIIC
(0.15 nM), and immunoprecipitated with anti-CNA3, anti-CNC1,
or control IgG. The specificity of immunoprecipitation of
I-
-conotoxin MVIIC receptors with anti-CNA3 was
tested by peptide block. Anti-CNA3 was preincubated with 20 µM CNA3 peptide or with 20 µM CNC1 peptide, and
immunoprecipitation was carried out as described under
``Experimental Procedures.''
Displacement of specific binding of I-
-conotoxin MVIIC by unlabeled
-conotoxin
MVIIC was observed between 10 pM and 1 nM, with
half-maximal inhibition at approximately 100 pM (Fig. 2). The K
value of
approximately 100 pM is in agreement with that determined in
rat synaptosomal membranes (10-300 pM) (Hillyard et
al., 1992; Kristipati et al., 1994; Woppmann et
al., 1994).
-Agatoxin IVA, a 48-amino acid peptide toxin
from funnel web spider venom with no obvious similarity in sequence to
-conotoxin MVIIC, blocks P-type calcium currents with IC
of 1-2 nM (Mintz et al., 1992a) and
calcium currents at 100-300 nM
(Sather et al., 1993, Stea et al., 1994a). To assess
whether
-conotoxin MVIIC and
-agatoxin IVA might bind to
the same high affinity sites, the ability of
-agatoxin IVA to
displace high affinity binding of
-conotoxin MVIIC was tested.
Displacement of specific binding of
I-
-conotoxin
MVIIC to the
-conotoxin MVIIC receptor site with unlabeled
-agatoxin IVA occurred only at high concentration with
half-maximal inhibition at approximately 1-5 µM (Fig. 2). This result indicated that
-agatoxin IVA
can displace
-conotoxin MVIIC from its high affinity binding site
nonspecifically at high concentration, but not at concentrations at
which it inhibits calcium channels containing
.
Evidently,
-agatoxin IVA does not bind to the same receptor site
as
-conotoxin MVIIC in inhibiting class A calcium channels.
Figure 2:
Competitive inhibition of I-
-conotoxin MVIIC binding by unlabeled
-conotoxin MVIIC or
-agatoxin IVA. The indicated
concentrations of
-contoxin MVIIC (closedcircles) or
-agatoxin IVA (opencircles) were added to WGA samples with
I-
-conotoxin MVIIC (0.15 nM) and
immunoprecipitated with affinity-purified anti-CNA3 antibodies. Bound
I-
-conotoxin MVIIC was determined, and the values
were normalized to the specific binding observed in the absence of
unlabeled toxins (100%).
Figure 3:
Detection of with
affinity-purified anti-CNA3 antibodies by immunoblotting. Membrane
glycoprotein fractions were isolated from solubilized brain membranes
by WGA affinity chromatography, and calcium channels were concentrated
by adsorption to heparin agarose, extracted, and analyzed by SDS-PAGE
using a 5% acrylamide gel (lanes 1-3) or a 7% acrylamide
gel (lane4). Proteins were transferred onto a
nitrocellulose membrane, blocked, incubated with anti-CNA3 (lanes1, 2, and 4) or anti-CNC1 (lane3), incubated with horseradish peroxidase-protein A,
washed, and visualized with ECL reagent, as described under
``Experimental Procedures.'' Anti-CNA3 antibodies were
preincubated overnight on ice with 2 µM CNA3 peptide (lane2). The migration positions of
- and
-spectrin, myosin heavy chain,
-macroglobulin,
-galactosidase, and fructose-6-phosphate kinase are indicated at
the left side of the gel together with their molecular masses in
kDa.
The specificity of the interaction of
anti-CNA3 antibodies with these polypeptides was tested with the CNA3
peptide. After preincubation with CNA3 peptide at a concentration of 2
µM, no signal could be detected with anti-CNA3 antibody (Fig. 3, lane2). These specific bands were
only observed with affinity-purified anti-CNA3 antibodies;
affinity-purified anti-CNC1 antibodies against revealed distinct bands with apparent molecular mass values of
210 and 190 kDa in the parallel gel (Fig. 3, lane3), and antibodies directed against the other brain
calcium channels also recognized distinct polypeptides (data not
shown). Thus, the 220-, 190-, and 160-kDa polypeptides are distinct
size forms of
subunits. From these results, we
cannot exclude that one or more of the multiple size forms of
is created by in vitro proteolysis.
However, it seems unlikely because inclusion of high concentrations of
several protease inhibitors and careful handling of all instruments
including rotors to keep them at 0 °C did not alter the appearance
of multiple forms of
. In addition, results presented
below indicate that the 220- and 190-kDa forms contain the
NH
- and COOH-terminal sequences predicted from cDNA
sequence, indicating they have not been proteolytically cleaved. The
doublet band with an apparent molecular mass of 220 kDa appears smaller
than the deduced molecular mass of
from cDNA
sequence (250 kDa; Starr et al., 1991), but it may represent
the full-length form of
because
subunits migrate anomalously in SDS-PAGE (De Jongh et
al., 1991). For example, an apparently full-length form of
(predicted molecular mass of 250 kDa; Snutch et
al., 1991) migrated to a similar position in the same gel (Fig. 3, lanes1 and 3) (Hell et
al., 1993a).
We also examined the polypeptides in immunoprecipitation experiments (Fig. 4).
To isolate and detect
in immunoprecipitation, we
biotinylated the WGA-purified glycoprotein fraction, isolated
by double immunoprecipitation with affinity-purified
anti-CNA3 antibodies, and visualized
by the
streptavidin-biotin detection method (see ``Experimental
Procedures''). When samples were analyzed in 5% acrylamide
SDS-PAGE gels, anti-CNA3 antibody revealed at least two distinct
immunoreactive polypeptides, which correspond to 220- and 190-kDa forms
detected in immunoblotting (Fig. 4, lane1).
The 190-kDa polypeptide was the major form, whereas 220-kDa polypeptide
was a minor form and was often visualized as a doublet band as in
immunoblotting. Occasionally, an additional band with a molecular mass
of 160 kDa was weakly stained in immunoprecipitation experiments. The
160-kDa polypeptide may not be sufficient in quantity to be detected
consistently in double immunoprecipitation.
Figure 4:
Detection of with
anti-CNA3 antibodies by double immunoprecipitation. WGA glycoprotein
fractions were biotinylated with NHS-LC-biotin, and
polypeptides were purified by double
immunoprecipitation. Samples were extracted with SDS-sample buffer,
separated by SDS-PAGE, blotted, and incubated with
streptavidin-biotinylated horseradish peroxidase complex as described
under ``Experimental Procedures.'' Calcium channels were
purified by double immunoprecipitation with affinity-purified anti-CNA3 (lane1), with anti-CNA3 and anti-CP(1382-1400) (lanes 2-4), with anti-CNA1 and anti-CP(1382-1400) (lane5), or with rabbit control IgG (lane6), and visualized by ECL. Anti-CNA3 antibodies were
preincubated overnight on ice with 50 µM CNA3 peptide (lane3) or 50 µM CNC1 peptide as a
control of the specificity of the peptide block (lane4). Molecular marker proteins are indicated as in Fig. 3.
The immunoreactive
polypeptides of 220 and 190 kDa were specifically recognized by
affinity-purified anti-CNA3 antibodies in immunoprecipitation, since
preincubation with CNA3 peptide at 50 µM eliminated the
immunoprecipitation of polypeptides with anti-CNA3,
but the CNC1 peptide at the same concentration had no effect (Fig. 4, lanes3 and 4). In addition,
neither band was recognized when immunoprecipitated with nonspecific
antibodies (Fig. 4, lane6). These specific
bands of
polypeptide were only observed with
affinity-purified anti-CNA3 antibodies, whereas affinity-purified
antibodies against
revealed distinct bands with
apparent molecular masses of 230 and 210 kDa in parallel experiments
(data not shown).
Calcium channels are multisubunit complexes and
may interact with other cellular components such as cytoskeletal
proteins and synaptic vesicle proteins in immunoprecipitation.
Therefore, it is possible that the proteins immunoprecipitated by
anti-CNA3 antibodies under native conditions might be associated
proteins of similar size to the subunit rather than
the
subunit itself. To exclude other proteins from
the immunoprecipitates, double immunoprecipitation experiments were
performed under conditions that should completely dissociate the
calcium channel subunits and associated proteins.
Anti-CP(1382-1400), which recognizes a segment of the
subunit whose sequence is conserved in all calcium channel
subunits so far characterized, was used as a probe in
the second immunoprecipitation. The CP(1382-1400) sequence is
accessible to anti-CP(1382-1400) only after solubilization in
Triton X-100, which removes the
and
subunits
(Ahlijanian et al., 1991). Following the double
immunoprecipitation with anti-CNA3 and anti-CP(1382-1400)
antibodies, two immunoreactive bands corresponding in size to the 220-
and 190-kDa polypeptides were visualized (Fig. 4, lane1), indicating that these immunoreactive polypeptides are
subunits. Two forms of
polypeptides with apparent molecular masses of approximately 220
and 190 kDa were also recognized by the affinity-purified anti-CNA1
antibody, which is directed against a unique amino acid sequence in the
intracellular loop between domains II and III of
(residues 865-881) immediately on the
NH
-terminal side of the CNA3 sequence. Double
immunoprecipitation with anti-CNA1 and anti-CP(1382-1400)
antibodies revealed two immunoreactive bands with molecular masses of
220 and 190 kDa, and the 190-kDa polypeptide was the major form of
as detected with anti-CNA3 antibody (Fig. 4, lane5). These observations in immunoblotting and
immunoprecipitation experiments demonstrate that
subunits consist of at least two distinct polypeptides that are
specifically recognized by anti-CNA3 antibody, and that the 190-kDa
polypeptide is a major form of
whereas the 220-kDa
polypeptide is a minor form of this subunit.
Immunoblotting with anti-BI-Nt
antibodies revealed four immunoreactive bands with apparent molecular
masses of 220, 190, 160, and 95 kDa in a 5% acrylamide gel (Fig. 5, lane1). These immunoreactive
polypeptides were specifically detected with anti-BI-Nt antibodies,
since 0.2 µM of the BI-Nt peptide blocked the interaction
of anti-BI-Nt with the immunoreactive polypeptides (Fig. 5, lane2). The immunoreactive band of 190 kDa was the
major form, and the band at 220 kDa was a doublet as observed with
anti-CNA3. In other blots, we stripped the membrane used for
immunoblotting with anti-BI-Nt antibodies by incubation at 50 °C
for 30 min in Tris-HCl buffer (pH 6.7) containing 2% SDS and 20 mM dithiothreitol, and re-probed with anti-CNA3 antibodies.
Immunoreactive bands with molecular masses of 220, 190, and 160 kDa
detected with anti-BI-Nt or anti-CNA3 were identical (data not shown).
Anti-BI-1-Ct antibodies revealed an immunoreactive band with an
apparent molecular mass of 190 kDa, which was blocked by preincubation
with 2 µM BI-1-Ct peptide (Fig. 5, lanes3 and 4). Anti-BI-1-Ct antibodies did not detect
immunoreactive bands with molecular mass values of 220 or 160 kDa in
immunoblots, possibly because of insufficient quantity of these
polypeptides in situ. Thus, immunoblotting with anti-BI-Nt and
anti-BI-1-Ct shows that the 190-kDa form of polypeptide contains both the predicted NH
- and
COOH-terminal ends of the
subunits.
Figure 5:
Detection of with
anti-BI-Nt and anti-BI-1-Ct antibodies by immunoblotting. Membrane
glycoprotein fractions were isolated from solubilized brain membranes
by WGA affinity chromatography, and calcium channels were concentrated
by adsorption to heparin agarose, extracted, and analyzed by SDS-PAGE.
Immunoblotting was performed with anti-BI-Nt (lanes1 and 2) or with anti-BI-1-Ct antibodies (lanes3 and 4) as described before (Fig. 3). To
test for nonspecific labeling, anti-BI-Nt antibody was preincubated
overnight on ice with 0.2 µM BI-Nt peptide (lane2), and anti-BI-1-Ct was preincubated with 2 µM BI-1-Ct peptide (lane4). Molecular mass markers
are indicated in Fig. 3.
It is
possible that anti-BI-Nt or anti-BI-1-Ct may recognize different
immunoreactive polypeptides with equivalent molecular weights or may
recognize since the BI-Nt sequence is 54% identical
to the corresponding
sequence. To exclude this
possibility, we performed double immunoprecipitation with anti-CNA3
antibodies and either anti-BI-Nt or anti-BI-1-Ct antibodies (Fig. 6). We used anti-BI-Nt or anti-BI-1-Ct antibodies for the
first immunoprecipitation and followed with anti-CNA3 or
anti-CP(1382-1400) in the second immunoprecipitation. Double
immunoprecipitation with anti-BI-Nt and anti-CP(1382-1400)
revealed two distinct immunoreactive polypeptides with molecular masses
of 220 and 190 kDa (Fig. 6, lane1), and
immunoprecipitation with anti-BI-1-Ct and anti-CP(1382-1400)
antibodies detected two forms of
polypeptide with
equivalent molecular weights (Fig. 6, lane4).
In double immunoprecipitation with anti-BI-Nt and anti-CNA3 antibodies,
polypeptides recognized by anti-BI-Nt were
specifically immunoprecipitated with anti-CNA3 antibody, since
preincubation of 50 µM CNA3 peptide blocked the
interaction of anti-CNA3 with
polypeptides (Fig. 6, lanes2 and 3). Similarly,
immunoprecipitation with anti-BI-1-Ct and anti-CNA3 detected
immunoreactive polypeptides of 220 and 190 kDa and was blocked by 50
µM CNA3 peptide (Fig. 6, lanes5 and 6). These results demonstrated that anti-CNA3,
anti-BI-Nt, and anti-BI-1-Ct antibodies recognized the same
immunoreactive
polypeptides with molecular mass
values of 220 and 190 kDa, and that 220- and 190-kDa forms of
have both the predicted NH
- and
COOH-terminal ends of
. These results indicate that
these isoforms of
do not result from
post-translational proteolytic processing, but may instead be products
of alternative RNA splicing.
Figure 6:
Recognition of
polypeptide in double immunoprecipitation by anti-CNA3, anti-BI-Nt, and
anti-BI-1-Ct antibodies. WGA glycoprotein fractions were biotinylated
with NHS-LC-biotin and purified by double immunoprecipitation with
anti-CNA3 antibodies. Samples were extracted with SDS-sample buffer,
separated by SDS-PAGE, blotted, and incubated with
streptavidin-biotinylated horseradish peroxidase complex as described
under ``Experimental Procedures.'' Double
immunoprecipitations were performed with anti-BI-Nt and
anti-CP(1382-1400) (lane1), with anti-BI-Nt
and anti-CNA3 (lanes2 and 3), with
anti-BI-1-Ct and anti-CP(1382-1400) (lane4),
or with anti-BI-1-Ct and anti-CNA3 antibodies (lanes5 and 6). Anti-CNA3 antibodies were preincubated overnight
on ice with 50 µM CNA3 peptide (lanes3 and 6) in the second immunoprecipitation. Note that the
blot was overexposed to demonstrate the immunoreactive band at 220 kDa (lane4). Molecular markers are given in Fig. 3.
Figure 7:
Phosphorylation of the class A calcium
channel subunits by PKA, PKC, and PKG. Class A
calcium channel
subunits were purified from WGA
glycoprotein fractions by immunoprecipitation with anti-CNA3 (lanes1, 3, and 5), or with rabbit control
antibodies (lanes2, 4, and 6).
Immunoprecipitated
was phosphorylated with PKA (lanes1 and 2), PKC (lanes3 and 4), or PKG (lanes5 and 6)
and reprecipitated with anti-CP(1382-1400) antibodies as
described under ``Experimental Procedures.'' Molecular mass
markers are described in Fig. 3.
The 190-kDa form of was a substrate for
phosphorylation by PKC and PKG (Fig. 7, lanes3 and 5). Control rabbit IgG was ineffective in
precipitating the 190-kDa polypeptides phosphorylated by these enzymes
confirming the identification of
(Fig. 7, lanes4 and 6). The 220-kDa form of
could not be visualized as a phosphorylated
polypeptide by PKC or PKG. However, we cannot exclude the possibility
that the 220-kDa form of
is also a substrate for
these kinases, since 220-kDa polypeptide could be present in
insufficient quantity for detection of phosphorylation by these
enzymes. Nevertheless, the results show that PKA preferentially
phosphorylates the 220-kDa form of
whereas PKC and
PKG preferentially phosphorylate the 190-kDa form.
Our results are closely correlated with covalent
cross-linking experiments identifying the polypeptides that bind
-conotoxin MVIIC (Woppmann et al., 1994). Polypeptides
with apparent molecular masses of 220, 170, 150, and 140 kDa were
observed. Although one or more of these bands may represent
subunits of class A channels or other associated proteins, it
seems most likely that the bands of 220 and 170 kDa correspond to the
220- and 190-kDa isoforms of
.
subunits have been suggested to be components of both P-type and
Q-type calcium channels.
is localized in high
density in the cell bodies and dendrites of cerebellar Purkinje cells
where P-type calcium currents are recorded, as well as in the cell
bodies and nerve terminals of cerebellar granule cells where Q-type
calcium currents are recorded (Westenbroek et al., 1995).
Coexpression of
with various calcium channel
subunits results in modulation of the amplitude, time course, and the
voltage-dependent properties of the
calcium currents
(Mori et al., 1991; Sather et al., 1993; Stea et
al., 1994a; Soong et al., 1994; De Waard et al.,
1994).
calcium currents expressed in Xenopus oocytes inactivate more rapidly than native P-type calcium
channels, but coexpression of the rbA-I or rbA-II isoforms of the
subunit with a
subunit (rbA-I with
or rbA-II with
) in Xenopus oocytes gives currents with much slower inactivation like a P-type
calcium channel (Stea et al., 1994a; Soong et al.,
1994). However, the sensitivity of
to
-conotoxin MVIIC and
-agatoxin IVA is not significantly
affected in these coexpression studies. These findings suggest that
pharmacological and physiological differences between Q-type and P-type
calcium channels may be due to an unidentified isoform of
, which may result from alternative RNA splicing or
post-translational modifications or may result from assembly with other
auxiliary subunits of calcium channels.
The BI subunit cDNA
clones from rabbit brain (BI-1 and BI-2) encode Q-type calcium channels
when expressed in Xenopus oocytes (Mori et al., 1991;
Sather et al., 1993). Analysis of BI clones revealed multiple
isoforms differing by insertion/deletion of 349 amino acids in the loop
between domains II and III (residues 772-1,120) and 195 amino
acids in COOH-terminal region and by alternative expression of a
28-amino acid substitution in COOH-terminal region (Mori et
al., 1991). These differences, which apparently result from
alternative RNA splicing, can give rise to at least eight distinct
mRNAs encoding multiple size forms of
in rabbit
brain. The differences in size caused by either of these large
deletions would be sufficient to reduce the apparent size of the
subunit from 220 to 190 kDa. These findings suggest
the possibility that
subunits in both rat and rabbit
contain multiple splicing cassettes in the loop between domains II and
III and in the COOH-terminal region and that the two size forms that we
have observed in our biochemical experiments are derived from these
alternative splicing events. Because the known cDNAs could encode
multiple
subunits with a size of approximately 190
kDa, it is possible that multiple
isoforms are
contained within the protein bands present in this region of the gel.
In addition, because the immunostaining with anti-BI-Ct was weaker than
with anti-BI-Nt, it is possible that this band also contains
subunits that have been truncated at the COOH
terminus by proteolytic processing.
In immunoblotting experiments,
affinity-purified anti-CNA3 and anti-BI-Nt antibodies specifically
identified an additional immunoreactive band of with
an apparent molecular mass of 160 kDa ( Fig. 3and Fig. 5). The biochemical properties of the 160-kDa form of
polypeptide could not be extensively characterized,
since this form was not consistently detected in double
immunoprecipitation experiments. However, our results show that the
160-kDa polypeptide contains both the CNA3 sequence and the BI-Nt
sequence. It may be an additional spliced isoform of
, or a proteolytic product of the longer forms of
polypeptide, which has a cleaved COOH terminus.
P-type and/or Q-type calcium channels are modulated by
GTP-binding proteins (G protein) and protein phosphorylation. P-type
channels in Purkinje neurons and spinal cord interneurons are inhibited
by -aminobutyric acid (GABA) through GABA
receptor
activation and this inhibition is mediated through G proteins (Mintz
and Bean, 1993). On the other hand, in hippocampal CA3 pyramidal
neurons, P-type calcium channels are potentiated by adenosine through
A
receptor activation (Mogul et al., 1993). This
potentiation involves a PKA-dependent process (Mogul et al.,
1993). Phosphorylation by second messenger-activated protein kinases is
a well known pathway for functional modulation of neuronal calcium
channels. Injection of cerebellar mRNA into Xenopus oocytes
leads to the expression of a single type of voltage-gated calcium
channels similar to P-type channels, and this calcium current is
enhanced by activators of PKA and PKC (Fournier et al., 1993a,
1993b). In contrast, I
of
channels
coexpressed with
subunit in Xenopus oocyte is not
affected by the activation of PKC (Stea et al., 1994b).
Whereas the functional effects of phosphorylation of P-type and Q-type
calcium channel are still incompletely described, our results provide
the first evidence that class A calcium channel
subunits are substrates for phosphorylation by PKA, PKC, and PKG,
and indicate that the different
subunit size forms
may be differentially phosphorylated and differentially regulated.
Further work is required to determine whether different isoforms of the
class A calcium channels are differentially regulated by PKA, PKC, and
PKG in vivo and to evaluate the physiological effect of
phosphorylation on
channel function.