(Received for publication, February 6, 1997)
From the Institut für Biochemische Pharmakologie, Peter-Mayrstrasse 1, A-6020 Innsbruck, Austria and the § Max-Delbrück Centrum für Molekulare Medizin, D-13125 Berlin, Federal Republic of Germany
Heterologous expression studies have shown that
the activity of voltage-gated Ca2+ channels is
regulated by their subunits in a
subunit isoform-specific manner. In this study we therefore investigated if one or several
subunit isoforms associate with L-type Ca2+ channels in
different regions of mammalian brain.
All four subunit isoforms (
1b,
2,
3, and
4) are
expressed in cerebral cortex as shown in immunoblots.
Immunoprecipitation of (+)-[3H]isradipine-labeled
L-type channels revealed that the majority of
subunit-associated L-type channels was associated with
3 (42 ± 8%) and
4 (42 ± 7%) subunits, whereas
1b and
2 were
present in a smaller fraction of channel complexes.
3 and
4 were
also the major L-type channel
subunits in hippocampus. In
cerebellum
1b,
2, and
3 but not
4 subunits were expressed
at lower levels than in cortex. Accordingly,
4 was the most
prominent
subunit in cerebellar L-type channels. This
subunit
composition was very similar to the one determined for
125I-
-conotoxin-GVIA-labeled N-type and
125I-
-conotoxin-MVIIC-labeled P/Q-type channel complexes
in cerebral cortex and cerebellum.
Our data show that all four subunit isoforms associate with L-type
Ca2+ channels in mammalian brain. This
subunit
heterogeneity may play an important role for the fine tuning of L-type
channel function and modulation in neurons.
Voltage-gated Ca2+ channels control the
depolarization-induced influx of extracellular Ca2+ into
neurons and other electrically excitable cells. They exist as
hetero-oligomeric complexes of different subunits (1,
2-
, and
). Different types of neuronal Ca2+ channels (termed L-,
N-, P-, Q-, and R-type; 1) are discriminated by biophysical and
pharmacological criteria (for reviews see Refs. 2-5). N- and P/Q-type
channels are blocked by peptide toxins (
-CTx1-GVIA and
-CTx-MVIIC or
-agatoxin-IVA, respectively), whereas L-type channels are modulated
by drugs, such as dihydropyridines (6). These channel types are
differentially distributed in the brain and even within a neuron (7,
8). Thereby they serve different physiological functions. N- and
P/Q-type channels are abundant in nerve terminals and control
Ca2+-dependent neurotransmitter release (3).
L-type channels are localized mainly on neuronal cell somata and
proximal dendrites where they may control
Ca2+-dependent modulatory processes and
excitation-transcription coupling (9).
The above Ca2+ channel types consist of different 1
subunit isoforms (class A-E) that also form their drug or toxin
binding domains and therefore determine their pharmacological
properties (1). In contrast, important biophysical and modulatory
properties, such as voltage-dependent gating (10, 11) and
channel modulation by G-proteins (12, 13) and kinases (14), are
determined not only by
1 but also by associated
2-
and
-subunits. Whereas only one
2-
isoform is known, four
different
subunit isoforms (
1-
4) are expressed in mammalian
brain (15, 16). Heterologous expression studies revealed that
subunits can affect
1 function in a
subunit isoform-specific
manner. For example, Ca2+ currents carried by
1A,
1E,
and
1C inactivate faster with coexpressed
3 than with
2 (14,
17, 18) subunits.
1,
3, and
4, but not
2, are permissive
for voltage-dependent facilitation of Ca2+
channels formed by
1C (19).
3 and
1 subunits confer slightly different pharmacological properties to L-type channels (20). Therefore
subunit heterogeneity could participate in the fine-tuning of
channel function. However, it is unclear if only one or several
subunit isoforms associate with these channels in mammalian brain. So
far only the
subunit composition of L-type Ca2+
channels in skeletal muscle has been studied. In this tissue exclusively
1a subunits are associated with the channel complex (15,
21).
Biochemical evidence for subunit heterogeneity in mammalian brain
has recently been provided for N-type and P/Q-type channels (22, 23).
Multiple
subunit isoforms were found to be associated to different
extents with both channel types after extraction from whole rabbit
brain.
Here we report that subunit heterogeneity also exists within
neuronal L-type channels. We found that regional differences in the
subunit expression pattern affect
subunit composition in different
regions of mammalian brain.
A preliminary report of our findings has appeared previously (24).
Reagents were obtained from the following
sources: 125I--CTx-GVIA, 125I-
-CTx-MVIIC
(2200 Ci/mmol), and (+)-[3H]isradipine from DuPont NEN
(Vienna, Austria); unlabeled
-CTx-GVIA from Sigma (Vienna, Austria);
unlabeled
-CTx-MVIIC from Saxon Biochemicals (Hannover, Germany);
prestained molecular weight markers from Bio-Rad (Vienna, Austria);
glutathione-Sepharose from Pharmacia (Vienna, Austria); Protein
A-Sepharose from Sigma; calpain inhibitors I and II from Boehringer
Mannheim (Vienna, Austria); all other protease inhibitors from
Sigma.
For antibody production in
rabbits peptides were coupled to bovine serum albumin with
glutaraldehyde (25) or synthesized on a lysine branch (octavalent
NovaSyn PA resin, Novabiochem) for immunization. Anti-2 was
generated as described (26). For immunoblotting and
immunoprecipitation experiments, antibodies were purified by affinity
chromatography on Sepharose-4B derivatized with the antigenic peptide
(25). Antigenic epitopes comprised the following amino acids (residue
number is given according to the sequences in Ref. 27):
1b,
516-530;
2, 595-604;
3, 470-483;
4, 460-474. Anti-
com
was raised against residues 61-79 in
1a (28).
Membranes were prepared from guinea
pig or rabbit cortex, hippocampus, cerebellum, and heart muscle as
described (29). Brain regions were rapidly removed from rabbit or
guinea pig brains and immediately placed in ice-cold homogenization
buffer containing 0.02 M NaHCO3 and a protease
inhibitor mixture (2 mM EDTA, 0.2 mM PMSF, 0.5 mM benzamidine, 2 mM iodoacetamide, 1 µM pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin,
20 µg/ml calpain inhibitor I and II, 0.1 mg/ml trypsin inhibitor).
The tissues were then homogenized by 10-20 strokes in a Dounce
homogenizer, and microsomes were collected by centrifugation at
45,000 × g (10 min, 4 °C). Microsomes were then
washed three times with 50 mM Tris-HCl, pH 7.4 (37 °C),
containing the same protease inhibitor mix. Membranes were resuspended
in the same buffer at a protein concentration about 5 mg/ml and stored
at 80 °C until use.
Glutathione S-transferase (GST) and a
GST fusion protein with the 1 subunit interaction domain of the
1A subunit (AIDA) were prepared as described (21). All further steps
were carried out on ice or at 4 °C. Typically 20 mg of microsomal
protein isolated from rabbit or guinea pig brain regions were
solubilized in 9 ml of buffer A (50 mM Tris-HCl, pH 7.4, containing the protease inhibitors used for membrane preparation)
supplemented with 1% (w/v) CHAPS and 1 M NaCl according to
Ref. 21. 30-µl aliquots of glutathione-Sepharose equilibrated in
buffer B (buffer A containing 0.1% (w/v) CHAPS, 0.1 M
NaCl) were coupled with 10 µg of GST or GST-AIDA and washed three
times with the above buffer. Solubilized membranes were diluted 10-fold
in buffer A, and 4 ml were mixed with the coupled glutathione-Sepharose
beads for 4 h or overnight. The beads were washed three times with
1.5 ml of buffer B, mixed with SDS-polyacrylamide gel electrophoresis
sample buffer (15 min, 56 °C or 3 min, 95 °C), and the eluted
protein separated on 10% polyacrylamide gels.
Immunoblot experiments were carried out as described (30). Prestained molecular weight markers (Bio-Rad) were run on the same gels. The apparent molecular masses of each batch were provided by the supplier.
Solubilization and ImmunoprecipitationMembrane-bound
channels were prelabeled with (+)-[3H]isradipine (1-2
nM) for 60 min at 37 °C in 50 mM Tris-HCl,
0.1 mM PMSF, 1 mM CaCl2. All
subsequent steps were carried out on ice or at 4 °C. Prelabeled
membranes were collected, solubilized on ice for 60 min in 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1% (w/v)
digitonin, 0.2 mM PMSF, 0.5 mM benzamidine, 2 mM iodoacetamide, 1 µM pepstatin A, and
nonsoluble proteins removed by centrifugation (45,000 × g, 60 min). The digitonin extracts were either used for
immunoprecipitation or were further affinity-purified by chromatography
on wheat germ agglutinin (WGA)-Sepharose (2 ml of packed resin for
5-20 mg of solubilized protein). Channel-associated activity was
eluted from the resin in equilibration buffer (50 mM
Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1 mM PMSF)
containing 6% (w/v) N-acetylglucosamine. Active fractions
were directly used in immunoprecipitation experiments or quickly frozen
in liquid nitrogen and stored at 80 °C until use.
For immunoprecipitation of labeled channels, affinity-purified
antibodies were coupled to Protein A-Sepharose. The Protein A-Sepharose-antibody complex was washed three times with 1.5 ml of
ice-cold RIA buffer (solubilization buffer containing 0.1% digitonin)
and then incubated for 12-16 h at 4 °C with 0.15-0.55 ml of
solubilized extracts (prelabeled N- and P/Q-type channels) or
WGA-Sepharose-purified channel preparations (L-type channels). Unbound
radioactivity was removed by four 1.5-ml washes with RIA buffer. Bound
radioactivity was determined by liquid scintillation ((+)-[3H]isradipine) or gamma-counting
(125I--CTx-GVIA, 125I-
-CTx-MVIIC).
High affinity binding of (+)-[3H]isradipine to solubilized L-type Ca2+ channels was determined using a filtration assay as described (29). This assay underestimated the total specific (+)-[3H]isradipine binding activity by about 20%. This was taken into account to calculate the binding activity employed for immunoprecipitation assays.
StatisticsData are given as means ± S.D. for the indicated number of experiments.
To investigate the association of all known subunit
isoforms with neuronal voltage-gated L-type Ca2+ channels
in mammalian brain, we raised anti-peptide antibodies against unique
sequences of 1b,
2,
3, and
4 subunits. In addition, an
antibody against an epitope highly conserved in all
subunit isoforms (anti-
com) was generated. We used these antibodies to determine their association with neuronal Ca2+ channels
solubilized from rabbit or guinea pig cerebral cortex, hippocampus, and
cerebellum membranes in immunoprecipitation experiments. Their
expression in these brain regions was analyzed in Western blots.
To determine their relative expression densities the four subunit
isoforms were extracted with CHAPS from microsomes prepared from brain
(
1b,
2,
3,
4) or, for control purposes, from skeletal muscle (
1a). Extracts were affinity-purified on GST-AIDA-Sepharose (21) in the presence of protease inhibitors as described under "Experimental Procedures." As shown in Fig. 1 for
skeletal muscle
1a (anti-
com staining) and neuronal
3 and
4
subunits, the enrichment of
subunit immunoreactivity was specific
and absent when only GST was used as the affinity matrix (Fig. 1,
lanes 4-6). In
subunit preparations from rabbit
cerebral cortex anti-
com specifically recognized a 63 ± 3/67 ± 3-kDa doublet and a 88 ± 4-kDa band (Fig.
2) (n
4). A ~33-kDa band was also
stained to a variable extent by
com as well as all the other
antibodies and corresponded to the GST-AIDA polypeptide present at
relatively high amounts (10 µg) in the
subunit preparations.
To assign the com-stained bands to individual
subunit isoforms,
samples separated on the same gel were stained with isoform-specific antibodies. The 88-kDa band was composed of anti-
1b (Fig.
2A) and anti-
2 staining (Fig. 2B). In
contrast, bands stained by anti-
4 and anti-
3 accounted for the
com immunoreactivity in the 63/67-kDa doublet. The majority of
anti-
3 immunoreactivity was associated with the larger 67-kDa
com
band, whereas anti-
4 recognized both bands of the doublet to a
variable extent (Figs. 1, 2, 3).
subunit isoform staining was specific. It was completely suppressed
in the presence of 1 µM of the respective antigenic peptides (not shown). As expected, only
com but not the
isoform-selective antibodies specifically recognized
1a extracted
from partially purified skeletal muscle T-tubule membranes (Fig.
2A).
com staining in rabbit heart represented
2
immunoreactivity (Fig. 2B) suggesting that other isoforms
are absent or expressed at much lower levels in this tissue.
The same bands were also present in hippocampus (Fig. 2) and cerebellum
extracts (Fig. 3). The relative abundance of the 88-kDa band was lowest in cerebellum because 1b and
2 expression density was lower in this region as compared with cerebral cortex (Fig. 2).
When similar amounts of solubilized membrane protein from cerebral
cortex and cerebellum were subjected to
subunit isolation and
Western blotting (Fig. 3) similar
com staining intensity was found
for the 63/67-kDa doublet.
3-specific immunoreactivity was less
abundant in cerebellum, whereas
4 was expressed at similar densities
as in cerebral cortex (Fig. 3).
Taken together, the com staining pattern in mammalian brain can be
explained by the presence of all four
subunit isoforms which are
expressed in a region-specific pattern.
After having
established the specificity of our antibodies, we investigated if
L-type Ca2+ channels are associated with only one or
several subunit isoforms and if
subunit association varies in
different brain regions. We reversibly labeled neuronal L-type
Ca2+ channels complexes in cerebral cortex, hippocampus,
and cerebellum membranes with the L-type Ca2+
channel-selective ligand (+)-[3H]isradipine and
solubilized them in buffer containing 1% (w/v) digitonin. In cerebral
cortex and hippocampus 74 ± 9% (n = 4) and
91 ± 22% (n = 4) of the solubilized
(+)-[3H]isradipine labeling was immunoprecipitated with
saturating concentrations of an antibody directed against
1C
indicating that binding was associated with L-type channel complexes.
61 ± 18% (n = 5) and 80 ± 31%
(n = 4) of the labeled L-type channels were
immunoprecipitated by
com. Therefore, most of the L-type channel
complexes are associated with a
subunit which is accessible for
com under nondenaturating conditions.
Immunoprecipitation experiments with the isoform-selective antibodies
revealed the association of more than one isoform with the channel
complex. Affinity-purified anti-
3 and anti-
4 antibodies each
immunoprecipitated 42% of the radioactivity recognized by anti-
com
(Fig. 4A). Smaller fractions were bound by
anti-
1b and anti-
2 (Fig. 4A). Together our
subunit-specific antibodies accounted for all (118%)
com
immunoprecipitable radioactivity in cerebral cortex.
Immunoprecipitation by these antibodies was saturable (see Fig.
5C). The nonspecific background signal
observed with the same concentrations of control rabbit immunoglobulin
was less than 10% (n > 3) of the radioactivity
recognized by anti-bcom. In control experiments only com, but none
of the isoform-specific antibodies, immunoprecipitated
(+)-[3H]isradipine-labeled L-type channels extracted from
rabbit skeletal muscle (Fig. 4B), which are exclusively
associated with
1a (21).
A similar subunit composition was observed in hippocampus (Fig.
4C). In the cerebellum only
4 accounted for a large
portion of L-type channel-associated
subunits (Fig. 4D).
Immunoprecipitation by
3 antibodies was less pronounced than in
cerebral cortex (Fig. 4D). This is in good agreement with
the lower relative abundance of
3 in this region (Fig. 3).
Immunoprecipitation by
1b and
2 was difficult to detect in
cerebellum (Fig. 4D) representing less than 10% of the
channels immunoprecipitated by anti-
com.
Together the isoform-selective antibodies accounted for most but not
all of the subunit-associated radioactivity in hippocampus (70%)
and cerebellum (66%).
Next we tested if the subunit
composition of L-type Ca2+ channels resembles the subunit
composition of N- and P/Q-type Ca2+ channels in these
regions (22, 23). For N- and P/Q-type channels it has been investigated
before in digitonin extracts of whole brain membranes, but data on
individual brain regions are unavailable. We therefore also subjected
125I-
-CTx-GVIA- and
125I-
-CTx-MVIIC-labeled channel complexes extracted from
cerebral cortex and cerebellum to immunoprecipitation with our
antibodies. We have previously shown that under our experimental
conditions saturable high affinity 125I-
-CTx-GVIA and
125I-
-CTx-MVIIC binding occurs selectively to N-type and
P/Q-type Ca2+ channels, respectively, with dissociation
constants in the subpicomolar range (31).
In cerebral cortex and cerebellum saturating concentrations of
anti-com recognized 85 ± 23% (n = 5) and
84 ± 13 (n = 4) of channels associated with
125I-
-CTx-GVIA binding activity, respectively. The
immunoprecipitation profile was very similar to L-type channels (Fig.
5, A and B).
3 and
4 subunits together
immunoprecipitated >80% of
com immunoprecipitable 125I-
-CTx-GVIA binding in a saturable manner (Fig.
5C). As with L-type channels, a smaller fraction of N-type
channel binding was associated with
1b and
2. In cerebellum again
only
4 antibodies recognized substantial portions of N-type channel
activity (Fig. 5B). Similar results as described for N-type
and L-type channels were also obtained for
125I-
-CTx-MVIIC-labeled P/Q-type channels in cerebral
cortex (not shown). In cerebellum only
4 antibodies recognized
significant amounts of 125I-
-CTx-MVIIC-labeled P/Q-type
channels (43 ± 18.5%, n = 3).
Isoform-selective antibodies completely accounted for the N-type
(105%, Fig. 5A) and P/Q-type (>85%, not shown) channel
binding recognized by anti-com in cerebral cortex but only for
40-50% in cerebellum. As for L-type channels this difference cannot
be attributed to differences in membrane preparation because it was also found when the respective brain regions were isolated from the
same animals in the same buffer and carried through the whole solubilization and immunoprecipitation procedure in parallel. It is
therefore possible that in hippocampus and cerebellum
immunoprecipitation by one or several of our antibodies was
underestimated. At present we do not know if this is due to the
expression of a yet uncharacterized
subunit isoform, which is
immunoprecipitated by
com but none of the other antibodies, or due
to region-specific differences in proteolysis. C-terminal proteolysis
could remove the C-terminal epitopes of our isoform-specific
antibodies. However, we have obtained no evidence for extensive
proteolytic breakdown of
subunits in immunoblots with our
com
antibody, which recognizes an epitope located near the N terminus of
the
subunits.
The major findings of our study are as follows. 1) All
known subunit isoforms participate in the formation of neuronal
L-type Ca2+ channels in mammalian brain. 2)
3 and
4
subunits are most often found as part of the neuronal L-type channel
complexes. 3) The fractional contribution of a particular
subunit
isoform for channel formation varies among different brain regions. 4)
The
subunit composition and regional differences are very similar to N- and P/Q-type channels in cerebral cortex and cerebellum.
This similarity of the subunit composition between L-type channels
and N- as well as P/Q-type channels is interesting because the
subcellular distribution of L-type channels in neurons differs significantly from the distribution of N- and P/Q-type channels. L-type
1C and
1D subunits are predominantly found on the cell soma and
proximal dendrites, whereas N-type
1B and P/Q-type
1A are also
found along the length of dendrites and in presynaptic terminals (8,
32). Despite these differences in neuronal targeting, these channel
types do not show major differences with respect to their
subunit
composition. Obviously different
subunit isoforms can be targeted
to different regions of a neuron.
Both 1C and
1D subunits participate in the formation of L-type
Ca2+ channels in mammalian brain. We have made no attempts
to determine if differences exist between the two L-type channels with
respect to their
subunit composition. The fraction of channels
associated with
1D is small (not more than 9-26% in hippocampus
and cerebral cortex as revealed by our immunoprecipitation experiments
with
1C; see also Ref, 33) and therefore complicates such an
analysis. We cannot exclude the possibility that
1b and
2, which
are found only in a minor fraction of channels, are selectively
associated only with
1D. However, based on our finding that
1C is
associated with the majority of labeled channels in cerebral cortex and
hippocampus,
-subunit heterogeneity must exist within class C L-type
channels in these regions.
subunits strongly affect the functional properties
of the pore-forming
1 subunits of L-type (and non L-type) channels. As shown by heterologous coexpression in Xenopus oocytes and
mammalian cells,
subunits affect channel gating (10, 11),
modulation by G-proteins (12, 13), and phosphorylation (14) as well as
Ca2+ and drug (34, 35) interaction with L-type
1
subunits. Such studies also revealed that different
isoforms are
able to confer different channel properties. For example,
3 confers
a more rapid inactivation to currents mediated by
1C (17) than does
2.
isoform-specific effects on channel inactivation were also
observed for non-L-type Ca2+ channel
1 subunits (11, 14,
18). Only
1,
3, and
4, but not
2, support
voltage-dependent facilitation of
1C-mediated Ca2+ currents (19). Similarly, small differences in the
sensitivity of the channel to the Ca2+ antagonist
mibefradil and the modulation by protein kinase C were observed when
different
subunits form part of the channel complex (14, 20). We
now provide direct biochemical evidence that indeed different
subunits participate in the formation of neuronal L-type channels
suggesting that these isoform-selective effects contribute to L-type
Ca2+ channel plasticity in mammalian brain.
subunits
could be involved in the fine tuning of L-type channel function in a
region-specific manner. Based on our findings future coexpression
studies should preferentially focus on the comparison of the properties
of L-type channels containing
3 or
4 subunits, because these
isoforms seem to be present in the majority of
dihydropyridine-sensitive L-type channels in cortex, hippocampus, and
cerebellum.
Taken together our data demonstrate that, like in other neuronal
Ca2+ channel types, several subunit isoforms contribute
to the formation of neuronal L-type channels. Further studies must
focus on the physiological and pathophysiological consequences of this
heterogeneity and investigate if changes in
subunit expression
could account for changes in L-type Ca2+ channel function
also under pathophysiological conditions, such as neurodegeneration,
cerebral ischemia, or aging (36).
We thank Dr. H. Glossmann for continuous support.