From the Department of Physiology and Biophysics,
Case Western Reserve University School of Medicine,
Cleveland, Ohio 44106-4970, § Max Delbruck Center,
Robert-Roessle-Strasse 10, 13122 Berlin, Germany, and
¶ ABL-Basic Research Program, NCI-Frederick Cancer Research and
Development Center, Frederick, Maryland 21702
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ABSTRACT |
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Calcium functions as an essential
second messenger during neuronal development and synapse acquisition.
Voltage-dependent calcium channels (VDCC), which are
critical to these processes, are heteromultimeric complexes composed of
1,
2/
, and
subunits.
subunits function to direct the VDCC complex to the plasma membrane as
well as regulate its channel properties. The importance of
to
neuronal functioning was recently underscored by the identification of
a truncated
4 isoform in the epileptic mouse lethargic
(lh) (Burgess, D. L., Jones, J. M., Meisler,
M. H., and Noebels, J. L. (1997) Cell 88, 385-392). The goal of our study was to investigate the role of
individual
isoforms (
1b,
2,
3, and
4) in the assembly
of N-type VDCC during rat brain development. By using quantitative
Western blot analysis with anti-
1B-directed antibodies and [125I-Tyr22]
-conotoxin GVIA
(125I-CTX) radioligand binding assays, we observed that
only a small fraction of the total
1B protein present in
embryonic and early postnatal brain expressed high affinity
125I-CTX-binding sites. These results suggested that
subsequent maturation of
1B or its assembly with
auxiliary subunits was required to exhibit high affinity
125I-CTX binding. The temporal pattern of expression of
subunits and their assembly with
1B indicated a
developmental pattern of expression of
isoforms:
1b increased
3-fold from P0 to adult,
4 increased 10-fold, and both
2 and
3
expression remained unchanged. As the
component of N-type VDCC
changed during postnatal development, we were able to identify both
immature and mature forms of N-type VDCC. At P2, the relative
contribution of
is
1b >
3
2, whereas at P14
and adult the distribution is
3 >
1b =
4. Although we observed no
4 associated with the
1B at P2,
4
accounted for 14 and 25% of total
1B/
subunit
complexes in P14 and adult, respectively. Thus, of the
isoforms
analyzed, only the
4 was assembled with the rat
1B to
form N-type VDCC with a time course that paralleled its level of
expression during rat brain development. These results suggest a
role for the
4 isoform in the assembly and maturation of the N-type
VDCC.
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INTRODUCTION |
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Ca2+ channels play important roles in neuronal development. Both VDCCs1 and calcium entry have been implicated in many processes of immature neurons including neurite outgrowth (1-3), neuronal migration (4), and axon and dendrite extension (5, 6). Calcium entry through VDCCs has been shown to be essential for sculpting neuromuscular synapses (7-10). Calcium channels have also been implicated in the initiation of developmental gene expression (11) and are necessary for spinal cord motoneuron differentiation in rat (12).
In vitro studies indicate that functional VDCCs are composed
of three subunits: 1,
2/
, and
.
Multiple VDCCs (N-, P/Q-, L-, and R-type) are expressed in neuronal
tissues (13, 14). The
1 subunit, of which there are at
least six genes, resides in the membrane and forms the pore of the
channel (14). The
subunit, a putative hydrophilic protein, binds
the
1 (15) and acts to regulate channel gating and
kinetics (16, 17). Four
subunit genes have been identified, several
of which exist as splice variants (14). N-type VDCCs are localized to
the plasma membrane of neuronal processes (18) and are essential for
the generation of action potentials and the subsequent release of neurotransmitters (19, 20). Adult N-type as well as other neuronal
VDCCs are heterogeneous in their
subunit component (21-24).
Although the in vivo process which gives rise to the
heterogeneity in
1B/
subunit complexes is not
understood, it is anticipated to generate N-type VDCC with different
channel properties based upon in vitro co-expression studies
(25, 26).
Developing neurons offer the opportunity to investigate the underlying
trends that contribute to subunit diversity. Several families of
ligand-gated ion channels evidence developmental changes in their
subunit composition (27-29). Whereas studies have identified alterations in the expression of N-type and P/Q-type VDCC during synapse formation in cultured neurons (30-32), few reports have investigated the developmental or differentiation-dependent
expression of calcium channel subunits and their assembly (33, 34).
Interestingly, there have been reports of N-type VDCC in developing
cerebellar granule cells (35) and differentiated human neuroblastoma
SH-SY5Y cells (36) which are inhibited by -conotoxin GVIA (CTX) yet display two components of inactivation. The expression of N-type VDCC
with different channel properties is a possible mechanism for
controlling membrane excitability. As
subunits are known to
influence the time course of channel inactivation, the diversity in
N-type VDCC activity was suggested to reflect heterogeneity in the
subunit component. We undertook this study to test directly the
hypothesis that N-type VDCCs are differentially associated with
subunits during rat brain development. The objectives of this
investigation are 1) to identify possible trends in calcium channel
subunit expression by evaluating changes in the expression of
1B and
isoforms throughout postnatal development and
2) to identify possible "immature" and "mature" forms of the
N-type VDCC by characterizing the
subunit component of N-type VDCC at different stages of rat brain maturation.
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EXPERIMENTAL PROCEDURES |
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MaterialsUnless noted, all reagents were obtained
from Sigma. Calpain inhibitors I and II were obtained from Calbiochem. Enhanced chemiluminescence kit (ECL) was purchased from Amersham Pharmacia Biotech; unlabeled
-GVIA conotoxin was from Peninsula Laboratories; [125I-Tyr22]
-conotoxin GVIA
(specific activity 2200 Ci/mmol) and 125I-protein A
(specific activity 21.1 µCi/µg) were obtained from NEN Life Science
Products. 125I-IgG was obtained from ICN (specific activity
12.9 µCi/µg). Nitrocellulose membranes were obtained from
Schleicher & Schuell. All goat anti-mouse secondary antibodies were
from Boehringer Mannheim. Sulfolink columns were purchased from Pierce.
Bovine serum albumin was from U. S. Biochemical Corp. Hepes was from
Research Organics.
Preparation of Rat Homogenates and Membranes--
Embryonic
(E18), neonatal, and adult rats were euthanized and the brains removed
and immediately placed in 50 mM Hepes, pH 7.4, 1 mM EGTA plus protease inhibitors at a protein to volume ratio of 1.3 g/25 ml. The protease inhibitors were added from stock
solutions prepared as follows: phenylmethanesulfonyl fluoride (1/1000
dilution from 200 mM stock in ethanol), calpain inhibitors I and II (1/1000 dilution from 4 mg/ml stock), benzamidine (1/500 dilution of 200 mM stock), aprotinin (1/500 dilution from 1 mg/ml stock), leupeptin (1/500 dilution from 1 mg/ml stock), pepstatin (1/500 dilution from 1 mg/ml stock in Me2SO), and DTT
(1/1000 dilution from 1 M stock). The tissue was
homogenized with a Polytron for 10 s and centrifuged at 18,000 rpm
(48,000 × g) for 15 min. The membranes were
resuspended in 5 ml of 50 mM Hepes, pH 7.4, plus protease
inhibitors at a resulting protein concentration of 3-10 mg/ml. For
subsequent use in Western blot analysis, all samples were stored in
20 °C at concentrations of 2 mg/ml in sample buffer (5× sample
buffer: 325 mM Tris, pH 7.0, glycerol (25% v/v),
mercaptoethanol (25% v/v), SDS (10%)) in 100-µl aliquots. The
samples were not freeze-thawed.
Production of Anti-peptide Polyclonal Antibodies to VDCC Subunit Epitopes-- The peptide antigens were synthesized to include a unique cysteine residue to be used both in the unambiguous attachment of peptide to carrier protein (maleimide-derivatized keyhole limpet hemocyanin) and to the affinity column (Sulfolink). The peptides were synthesized, purified, and coupled to maleimide-activated keyhole limpet hemocyanin. The coupled peptide antigens were used in the production of polyclonal sera in rabbits under continued contractual agreement with Covance, Inc. The rabbits were bled twice per month (15-20 ml/bleed) and tested for production of specific antibodies after 4 weeks as described previously (34).
Anti-Preparation of Peptide Columns-- The concentration of free sulfhydryl group available in the peptide sample was quantified by Ellman's assay with cysteine as the standard used in the range of 20-200 nmol/assay. The free peptide (1.5-3 mg in 2-ml volume) was coupled to a Sulfolink column (Pierce), and excess reactive groups were coupled to cysteine according to the manufacturer's instructions. The coupling efficiency was 94-99% as determined by both dot blotting and Ellman's assay.
Affinity Purification of Anti-peptide Antibodies--
Crude
antisera were diluted with 3% BSA in TBS and incubated with peptide
column for 90 min at room temperature. The column was then washed with
25 ml of TBS and sequentially eluted with 3 ml of 30 mM
glycine, pH 5, and 3 ml of 80 mM glycine, pH 4. The
affinity purified antibodies were eluted from the column with 6 ml of
200 mM glycine, pH 2.5, with each 1 ml collected and
immediately neutralized by the addition of 50 µl of 1 M
Tris, pH 9.5. The protein concentrations of the fractions were
determined, and the peak fractions were pooled and dialyzed overnight
in the cold room against 4 liters of TBS. The following morning, the
sample was dialyzed for an additional 4 h, and the purified
antibody was aliquoted in 50-100-µl volumes and stored at 80 °C
without freeze-thawing. The peptide column was extensively washed with TBS and stored in TBS plus 0.05% azide.
Production of Anti-peptide Polyclonal Antibodies to VDCC Subunits--
Anti-
subunit "generic" antibodies (Ab CW24) were
raised to a highly conserved sequence (CESYTSRPSDSDVSLEEDRE) present in all
subunits cloned to date that is not implicated in either the
binding of
to the
1 subunit or the consensus sites
for protein phosphorylation or ATP-binding in the
4 subunit (39). This peptide was coupled via a unique CYS as described above.
Methods for 125I-CTX
Binding--
125I-CTX
([125I-Tyr22]-conotoxin GVIA (specific
activity 2000 Ci (81.4 TBq/mmol)) binding was assayed by published
procedures which use filtration over PEI-soaked glass fiber filters
(Whatman GF/B) to separate bound from unbound ligand in the presence of
bovine serum albumin (BSA). N-type VDCC fractions were routinely
screened at several protein concentrations to determine the linear
range for the binding. Individual assay tubes contained 100 µl of
representative N-type VDCC fractions diluted into 50 mM
Hepes, pH 7.4, 100 µl of 1% BSA (w/v), 100 µl of
125I-CTX stock solution diluted to correspond to
approximately 20,000 cpm 125I-CTX (or approximately 4.2 fmol), 100 µl of 500 nM stock solution of unlabeled CTX
(Peninsula Laboratories) or 50 mM Hepes, pH 7.4 buffer in 1 ml final volume. The samples were incubated at room temperature for 30 min, filtered over 0.5% PEI-soaked glass fiber filters, and rapidly
washed as described. The filters were counted for 1 min in Packard
Cobra autogamma counter. Scatchard analysis of 125I-CTX
binding to membranes was carried out under similar conditions with
protein assayed at dilutions that supported approximately 2,500 cpm of
specific 125I-CTX bound (1-200 µg/ml per assay). The
amount of protein present in each assay was as follows: adult, 2 µg;
P0, 25 µg; and E18 rat brain, 200 µg. Unlabeled CTX added was from
0.1 pM to 50 nM in the presence of constant
125I-CTX. Data presented are mean ± S.D. from three
determinations done in duplicate.
Immunoprecipitation of N-type VDCC-- The N-type VDCC was solubilized from P2 brain and P14 and adult rat forebrain as described previously (21) with the following modifications: the N-type VDCC from P2 brain and P14 forebrain were solubilized directly from membranes using 0.75% CHAPS. Following centrifugation, the solubilized preparations were assayed for 125I-CTX binding. Approximately 9,000-12,000 cpm of specific 125I-CTX receptor activity (200 µl of the solubilized preparation) was added to individual microcentrifuge tubes that contained 20,000-40,000 cpm 125I-CTX, 0.1% BSA, plus protease inhibitors in 50 mM Hepes, pH 7.4. Identical reactions were also carried out in the presence of 50 nM unlabeled CTX to determine nonspecific binding of 125I-CTX. Following a 30-min incubation at room temperature, antibody was added in a total volume of 100-200 µl of TBS and left to incubate at room temperature for 1 h. After this time, 50 µl of protein A-Sepharose 4B (final concentration of 0.6 mg/ml) was added to each sample and rotated in the cold room overnight. 125I-CTX binding to the soluble fraction was determined by directly filtering 1.1 ml of the sample through 0.5% PEI-soaked glass fiber filters. The pellets were washed 3 × with 1 ml of 50 mM Hepes/EGTA, and 50 µl of 2× sample buffer were added to the protein A beads. The samples were counted in a gamma counter for 1 min.
Quantification of VDCC Subunits by 125I-Protein A
Overlay or 125I-Goat Anti-rabbit
IgG--
125I-Protein A and 125I-goat
anti-rabbit IgG were diluted in 50 ml of 3% BSA in 1× TBS. Filters
previously blocked with 5% milk in 1× TBS and probed with primary
antibody were washed with 1× TBS for 15 min and then washed two
additional times for 5 min. The washed filters were incubated in 50 ml
of either the 125I-protein A, 3% BSA solution
(approximately 500 cpm/µl) or the 125I-IgG, 3% BSA
solution (approximately 30-50 cpm/µl) for 2 h at room
temperature with constant shaking. Following this incubation, the
125I solution was removed, and the filters were washed 3-5
times with TBS (5 min each). The approximate wash volume was 50 ml. The
filters were blotted with paper towels and exposed to film at
80 °C with the aid of intensifying screens. The position of the
antigen was determined relative to the exposed film, and the corresponding band on the filter was cut and counted. Slices that corresponded to nonspecific areas of the filter were also counted and
subtracted from the signal. The data were obtained from multiple determinations done in duplicate.
General Methods and Data Analysis-- The gels were transferred to nitrocellulose at 0.45 A for 17-22 h. The filter was incubated in 5% powdered milk in TBS + 0.01% sodium azide + 0.05% Tween and blocked for either 3 h at 37 °C with constant shaking or overnight at 4 °C in the cold room. The primary antibody was diluted in 3% BSA, 1 × TBS and incubated with the filter overnight at 4 °C. The filters were washed 3 times in TBS at room temperature. The secondary antibody was diluted to 1/10,000 in 3% BSA, 1× TBS, and the filter was incubated for 45 min at room temperature with constant shaking. The filters were washed as before. The antigen was visualized using ECL. Membrane protein and soluble protein were measured by the Pierce BCA assay. Bovine serum albumin was used as a standard in all cases, and all samples were normalized with respect to buffer and detergent composition. Gel electrophoresis was carried out on polyacrylamide gels according to standard procedures (45). Gel electrophoresis was carried out using a 4% stacking gel and a resolving gel of appropriate porosity (see figure legends) according to standard procedures. All samples were incubated with 5× SDS-PAGE sample buffer (325 mM Tris, pH 7.0, glycerol (25% v/v), mercaptoethanol (25% v/v), SDS (10%) without boiling. Staining of proteins in polyacrylamide gels was Coomassie Blue (0.05%), 50% methanol, 10% acetic acid. The results are expressed as mean ± S.D. Statistical analysis was evaluated by a paired t test or one-way analysis of variance with Tukey's or Dunnett's post hoc test. p values less than 0.05 were considered significant.
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RESULTS |
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Expression and Properties of the N-type 1B Subunit
during Rat Brain Development
Immunological Characterization of Rat
1B--
Anti-peptide antibodies to the II-III
intracellular loop of the rat
1B subunit were raised to
two distinct epitopes. The first antibody, Ab CW14, was raised to an
epitope present in all
1B subunits cloned to date (34).
The second antibody, Ab CW8, was raised to an epitope present only in
the rat
1B sequence (37). Both antibodies were analyzed
in parallel to characterize the structure of the endogenous
1B as it relates to the original rat
1B
cDNA clone (37).
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Developmental Expression of 1B Subunit in Rat
Brain--
The change in expression of
1B subunit
presented in Fig. 2 leads us to use Ab CW14 to evaluate the level of
expression of
1B in postnatal (P0-P14) rat brain and
adult forebrain homogenates. As shown in Fig.
3A, the level of expression of
1B increases during rat brain development. The results
of similar Western blots were quantified using 125I-IgG and
evidenced statistically significant increase in expression of
1B subunit throughout the period of postnatal
development (Fig. 3B).
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Characterization of N-type VDCC 125I-CTX Binding during Three Rat Developmental Stages-- We used radioligand binding assays of the peptide neurotoxin, 125I-CTX, to determine if there were developmental differences in either the density or affinity of 125I-CTX binding to N-type VDCC. Radioligand binding assays using 125I-CTX were carried out on homogenates from embryonic day 18 brain (E18), newborn rat brain (P0), and adult rat forebrain. The results of the pseudo-Scatchard analysis are presented in Table I. A comparison of the Bmax values indicates significantly less 125I-CTX binding at E18 (Bmax = 0.008 ± 0.002 pmol/mg) and P0 (Bmax = 0.05 ± 0.01 pmol/mg) versus adult rat forebrain (Bmax = 1.8 ± 0.8 pmol/mg). The high affinity 125I-CTX binding site diagnostic for the N-type VDCC is present throughout development (Kd of 11.7, 21.7, and 8.3 pM for 125I-CTX binding to adult rat forebrain, P0 rat brain and E18 rat brain, respectively).
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Expression and Assembly of VDCC Subunits during Rat Brain
Development
Expression of Subunit Isoforms during Rat Brain
Development--
The level of expression of calcium channel
subunits in developing rat brain was then analyzed to evaluate possible
changes in the pool of available
isoforms. Thus, we used Ab CW24,
an antibody raised to an epitope shown to be present in all
subunits cloned to date (34), to probe a Western blot of homogenates prepared from developing rat brains. These experiments revealed two
populations of
subunits that could be easily resolved by SDS-PAGE
as follows:
subunits with apparent molecular masses of >80 kDa
comprised of
1b and
2 isoforms, and smaller
subunits (65 kDa)
comprised of
3 and
4 isoforms (34). As shown in Fig. 4, direct comparison of these two
populations of
subunits indicated no statistically significant
change in the level of expression of the larger
subunits between P0
and adult, whereas the smaller
subunits evidenced a significant
3-fold increase in expression. It is important to note that the
histogram reflects the expression of the
1b +
2 and
3 +
4
as the individual bands could not be resolved adequately (Fig. 4,
B and C).
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Expression of Specific Subunit Isoforms during
Development--
Therefore, to determine accurately the time course
specific to each
subunit isoform, the experiment was repeated using
isoform-specific antibodies. The changes we observed in
isoform expression are indicative of the total pool of available
as we
carried out the analysis in whole brain homogenate rather than in
plasma membrane fractions or biochemically purified preparations of
VDCC. As shown in Fig. 5A, the
results indicate a statistically significant increase in the expression
of the
1b isoform detected in P0 through adult with the increase
commencing at the time of cerebellar maturation (P7). The
2 isoform
(Fig. 5B) and
3 isoform (Fig. 5C) were
expressed at constant levels. Interestingly, there is a 10-fold
increase in the level of expression in the
4 isoform in adult brain
compared with P0 that also commences at the time of cerebellar
maturation (P7) (Fig. 5D). The increase in expression of the
4 detected in the P7-P14 interval in these rat brain samples parallels the increased expression of the
4 mRNA in cerebellum as determined by in situ hybridization (48).
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Heterogeneity of 1B/
Complexes during Rat Brain
Development--
The N-type VDCC has been previously purified from rat
(21, 49, 50) and rabbit forebrain (51), where its density of expression
is 2.5-fold higher than in cerebellum or other brain regions (52).
Therefore, N-type VDCC were solubilized from P2 brain, P14, and adult
rat forebrain and immunoprecipitated with the anti-
1B
antibody Ab CW14, and the generic anti-
subunit antibody Ab CW24.
The N-type VDCC present at early stages of rat brain development (P2
and P14) can be quantitatively immunoprecipitated by antibodies to the
1B (Fig. 6). However, only
60-70% of all 125I-CTX binding can be immunoprecipitated
by the generic antibody reactive toward all
subunits (Ab CW24).
These data identify a statistically significant fraction of
1B-binding sites in immature brain that are not tightly
associated with a
subunit. In contrast, the N-type VDCC extracted
from adult rat can be quantitatively immunoprecipitated by both
anti-
1B and Ab CW24.
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DISCUSSION |
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In the past several years, investigators have struggled to make
physiological sense of the vast diversity of VDCC subunit isoforms
present in neural tissues. The dramatic changes that occur in calcium
conductances during neuronal maturation suggest an underlying and
equally dramatic change in the density and subtype of VDCC. Indeed, in
cultured cerebellar Purkinje cells, studies have demonstrated that
changes in calcium conductances were critical for neuronal maturation
(53). More recently, important changes were observed in the expression
and differential contribution of N-type and P/Q-type VDCC during
synapse formation in cultured neurons (30-32). Similarly, changes in
calcium channel currents were observed at different stages of
embryogenesis (54). These studies were among the first to suggest
regulation of 1 subunit expression as a possible
mechanism for establishing diversity in calcium signaling during
development.
In support of previous findings on the role of N-type VDCC in neuronal
development, we have demonstrated an increase in the expression of
1B during rat brain development (Figs. 2 and 3) which
does not correlate with the acquisition of 125I-CTX-binding
sites (Table I). The parallel increase in reactivity of Ab CW8 and Ab
CW14 throughout development indicates that the
1B
present in these samples contains two epitopes originally identified in
the rat
1B cDNA (37). Functionally different isoforms of N-type VDCC have been identified in rat sympathetic ganglia
(38) and embryonic (E17) tissues (55); however, these variants contain
both Ab CW14 and Ab CW8 epitopes. The report of splice variants in the
II-III loop of
1A subunit suggests caution in
dismissing the existence of additional
1B variants. It
is important to note that the region defined by Ab CW8 is coincident with a region of diversity in the
1A variants (56).
In this study we have described a population of 1B
detected in embryonic and P0 brain samples that does not support high affinity 125I-CTX binding (Table I). This property is
reminiscent of the unassembled
1B expressed in
heterologous systems in the absence of
2/
and
subunits (57). Also, studies on the developmental expression of the
sodium channel have noted the acquisition of high affinity
[3H]saxitoxin binding occurring in parallel with channel
assembly (58). Our results suggest that the acquisition of high
affinity CTX binding during rat brain development (Table I) occurs by a
mechanism that is similar to the sodium channel and reflects the
conversion of the pool of unassembled
1B (present at E18 and P0) to mature
1B that are assembled with component
2/
and
.
This is the first report to demonstrate regulation of the subunit
component of a specific VDCC (N-type) during neuronal development.
Previously, correlations in the localization and density of
1 and
isoform mRNA in adult, embryonic, and
postnatal rat brain predicted likely
1/
complexes
(48), but there were no conclusions reached in the case of the
1B/
complex. Another study concluded that there was
no evidence for subunit switching in developing hippocampus as they
targeted only the
1B/
3 heteromultimers for analysis
(59). In this study we have identified clear developmental trends in
1B/
composition from a population predominant in
1B +
1b complexes (P2) to a population comprised of
1B +
3 and
1B +
4 complexes in
mature rat brain.
The discrepancy between the fraction of 125I-CTX-labeled
N-type VDCC that could be identified in P2 and P14 by
anti-1B antibodies versus those identified by
anti-
antibodies (Fig. 6) suggests the presence of a
isoform
that is not identified by our pan-specific antibody nor recognized by
-specific antibodies. Alternatively, there may be some structural
lability in the physical coupling of
to the
1B in
early development. A conserved site on the intracellular I-II loop has
been identified in all
1 subunits that bind
(60,
61). In vitro studies have determined nanomolar affinity
between all
and the I-II loop interaction domain in a binding
reaction that is not affected by calcium or protein kinase C
phosphorylation (61). Recently, a second site that mediates
1-
interaction has been identified at the C terminus of the
1E,
1A, and
1B (62,
63); however, the affinity for
at this site has not yet been
determined. The occupancy of these two sites on
1B by
adds another dimension to
1B/
heterogeneity.
This age-dependent structural heterogeneity in N-type VDCC
is anticipated to have a functional counterpart based upon recombinant studies of isoforms co-expressed with the
1C (26,
64),
1C (65), and
1E (65). We anticipate
significant cellular consequences related to differential expression of
1B/
complexes as
is required for the assembly,
stabilization, and targeting of
1 (66-69) in addition
to influencing the kinetic and modulatory properties of
1 (25, 26, 64, 70-72). The magnitude and duration of
calcium entry via specific
1B +
subunit complexes
and their localization may contribute to distinct signals that regulate neurite extension, synapse formation, and growth cone collapse (7-10).
Differential modulation of the N-type VDCC by protein kinases is
another property that may result from the assembly of a specific with the
1B. The
1b (41) and
2 (25) isoforms
contain consensus sites for protein kinase A modification and no
consensus site for tyrosine kinases; conversely, the
3 isoforms
contain consensus sites for tyrosine kinases and no consensus site for protein kinase A (43). In the
4, both protein kinase A and tyrosine
kinases consensus sites are absent (39). The functional consequences of
assembling different isoforms of
subunit that act as substrates for
different protein kinases into the N-type
1B/
complex
suggest a possible mechanism for coupling specific intracellular
signaling pathways to N-type VDCC.
The report of changes in 1 and
mRNA levels
during development (48) and our results (Fig. 5) suggest differential
regulation of
isoform expression. The effect of neurotrophic agents
upon changes in calcium currents (73-75) and the specific expression of
1B and
isoforms have been reported (33, 34) in
established neurotypic cell lines. However, these in vitro
models pale in comparison to the complexity of developing rat brain.
The trends identified in this study require further scrutiny at the
cellular level to unravel the mechanisms that underlie both
expression and its association with
1.
We would like to address the question whether the assembly of with
an
1 subunit is reflective of specific assembly
processes or simply reflects the fractional contribution of a
isoform relative to the pool of total available
. As demonstrated by our results, with the exception of the
4 subunit, there is no straightforward relationship that emerges between the relative contribution of
1b,
2, and
3 to the pool of available
subunits (Fig. 5) and the contribution of that isoform to the assembled N-type VDCC (Fig. 7).
A comparison of the heterogeneity in 1B/
complexes
through development with changes in the pool of available
isoforms suggests several cellular strategies are at play which regulate
1B/
subunit assembly. The
1b subunit is detected
in P2 homogenate at a fraction of its maximal adult level of expression
(Fig. 5A); however, the relative amount of
1b associated
with the P2 N-type VDCC is similar to the adult N-type VDCC (Fig. 7).
These findings indicate a relative enrichment of the
1b in the P2
N-type VDCC complex relative to adult N-type VDCC. As previously shown,
the
2 subunit is detected throughout rat brain development (Fig. 5B), yet antibodies to the
2 isoform do not
immunoprecipitate 125I-CTX binding from P2 or P14 samples.
In the adult samples, less than 10% of all
125I-CTX-labeled N-type VDCC were immunoprecipitated by
anti-
2 antibody. These findings suggest the active exclusion of the
2 subunit from the N-type VDCC complex. Similar to
2 expression,
the
3 subunit is also expressed at a relatively constant level in
the interval between P0 and adult. However, in a manner similar to the
4, there is a statistically significant increase in its association with the
1B during development. It is very interesting
to note that the onset of increased
1b and
4 expression occurs at
the beginning of a well defined period of axonal outgrowth,
infiltration, and synapse formation in the rat neocortex which occurs
in the first 2 weeks of postnatal life (76-78).
Significantly, the 10-fold increase in the expression of the 4
isoform between P0 and adult and its parallel association with the
1B through development is in striking contrast to the other
isoforms and identifies a property unique to the
4.
Interestingly, there has been a report that demonstrates the importance
of the
4 isoform. Analyses of the mutation that underlies the mouse lethargic phenotype (lh/lh), a model of epilepsy
which does not exhibit any neurodegeneration or other neurohistological
abnormalities (79), have identified an insert in the
4 gene that
leads to a truncated gene product. Specifically, the truncation of the
4 subunit protein eliminates the
1-binding domain as
well as more than 60% of the protein (80). The study by Burgess
et al. (80) is significant as it is the first to implicate
VDCC auxiliary subunit as the basis for a neurological disease. The
co-localization of the
1B and
1A with the
4 isoform in normal rat forebrain and cerebellum (48) and the
identification of the
4 as a component of the adult P-type (33),
N-type VDCC (22, 23), and L-type VDCC (23) suggest important lines of
investigation toward understanding the role of the
4 truncation in
epileptic lh/lh mice. It will be of interest to examine if
the association of the
4 with the other VDCC during development also
occurs in parallel to its level of expression. Furthermore, the
epileptic phenotype of lh/lh mice that results as a
consequence of a defect in a single
subunit intimates a role for
the
4 isoform that cannot be complemented by the expression of the
other
isoforms. Although it is clear from our study that the
4
isoform is unique among
subunits in its magnitude of induction and
temporal pattern of expression, it would be premature to suggest that
it is the absence of the
4 isoform per se that gives rise
to the epileptic lh/lh phenotype. Alternatively, one
might consider that alterations in the level of expression of
full-length
4 in lh/lh mice may induce profound compensatory effects upon the regulation of expression of the other
isoforms.
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ACKNOWLEDGEMENTS |
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The HEK293 cell line stably expressing the human N-type VDCC was a generous gift from our colleagues at SIBIA Neuroscience, Inc. We gratefully acknowledge Yunsook Choi and Dr. Marinela Pacioaou for their expert technical assistance. We also thank Stefan Dubel for preparing the figures for publication and for helpful discussions. Dr. Gary Landreth and Dr. Lynn Landmesser are acknowledged for their reading of the manuscript and thoughtful comments and suggestions.
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FOOTNOTES |
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* This study was funded by the National Institute of Mental Health (to M. W. M.), Life Health Insurance Medical Research Fund (to M. W. M.), the American Heart Association (to M. W. M), the Deutsche Forschungsgemeinschaft (to H. H), and in part by the NCI, DHHS, National Institutes of Health. under contract with ABL (to T. D. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Established Investigator of the American Heart Association. To
whom reprint requests should be addressed: Dept. of Physiology and
Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970.
1
The abbreviations used are: VDCC,
voltage-dependent calcium channel(s); N-type VDCC,
-conotoxin-sensitive VDCC;
1B, 230-kDa subunit of the
N-type VDCC;
2/
, 160-kDa subunit of N-type VDCC;
1
through
4, 53-85-kDa subunits of N-type VDCC; E18, embryonic day
18; P0-P14, rats aged postnatal day 0-postnatal day 14;
125I-CTX,[125I-Tyr22]
-conotoxin
GVIA; BSA, bovine serum albumin; CHAPS,
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; ECL, enhanced chemiluminescence; PEI, polyethyleneimine; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; Ab,
antibody; BSA, bovine serum albumin.
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REFERENCES |
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