From the Howard Hughes Medical Institute, Departments
of Physiology and Biophysics and Neurology, University of Iowa,
Iowa City, Iowa 52242, the § Department of Physiology,
University of Wisconsin, Madison, Wisconsin 53706, and the
¶ Departments of Biochemistry and Molecular Biology and
Ophthalmology and Visual Sciences, University of Louisville School
of Medicine, Louisville, Kentucky 40202
Received for publication, August 23, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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Voltage-gated calcium channels mediate
excitationcontraction coupling in the skeletal muscle. Their
molecular composition, similar to neuronal channels, includes the
pore-forming The L-type voltage-gated calcium channels of the skeletal muscle
serve both as a voltage-gated calcium channel and as a voltage sensor
for excitation-contraction
(EC)1 coupling (1, 2). These
channels serve to couple depolarization to intracellular calcium
release via the ryanodine receptor. The sites of EC coupling in the
skeletal muscle are the triads, which are highly organized junctions
comprising the t-tubules and the underlying sarcoplasmic reticulum (3).
The voltage-gated calcium channels are localized predominantly in the
t-tubules in close association with the ryanodine receptor in the
sarcoplasmic reticulum (4, 5).
At the molecular level, the calcium channel is composed of the
pore-forming The The Our data show that the skeletal L-type channel is indeed maintained as
a complex in the To examine the subunit of the channel with which the Enrichment and Purification of the Skeletal Voltage-gated Calcium
Channels--
KCl microsomes were prepared from wild type and
Constructs and Generation of Adenoviruses--
All the
adenoviruses and cell expression constructs used in this study
expressed the protein under the control of the cytomegalovirus promoter. The cDNAs of interest were subcloned into the
pAd5CMVK-NpA vector and used for generation of the adenovirus by
standard homologous recombination techniques. The recombinant viruses
were then purified using standard techniques. The Gene Transfer Vector
Core at the University of Iowa, Iowa City, IA, generated all
adenoviruses in this study. In some cases, the cDNAs in the
adenovirus vectors were used for cell expression studies.
Injection of Adenoviruses into Skeletal Muscle of the
Sucrose Gradient Fractionation--
Wheat germ
agglutinin-enriched material or KCl microsomes that were solubilized as
described in the methods for the purification of the channel complex
were concentrated and loaded on 5-30% linear sucrose gradients with
0.1% digitonin and 0.5 M NaCl. The gradients were
centrifuged for 90 min at 50,000 rpm in a 65.2 Ti rotor (Beckman Instruments). 800-µl fractions were collected from the top from each tube.
SDS-PAGE and Immunoblotting--
Samples were subjected to
SDS-PAGE on gradient gels and transferred to polyvinylidene difluoride
membranes for immunoblotting. The membranes were blocked and incubated
with the primary antibody, followed by secondary antibody and detected
by enhanced chemiluminescence.
Antibodies--
The antibodies used in this study have been
described previously, IID5E1, IIF7 ( Immunofluorescence Analysis--
Tissue samples
(quadriceps) were frozen and processed for immunohistochemistry as
described previously (26). The sections were labeled with a rabbit
polyclonal antibody to Primary Cultures and cDNA Transfection--
Primary cultures
were prepared from enzyme-digested hind limbs of late-gestation (E18)
Whole-cell Voltage Clamp and Solutions--
Cells were
voltage-clamped ~48 h after transfection. Transfected cells revealed
by CD8 beads were voltage-clamped with an Axopatch 200B amplifier (Axon
Instruments, CA) and a Digidata 1200 (Axon) pulse generator and
digitizer. Linear capacitance, leak currents, and effective series
resistance were compensated with the amplifier circuit. The voltage
dependence of the Ca2+ conductance was fitted according to
a Boltzmann distribution, A = Amax/(1 + exp( Analysis of mdg and Wild Type Muscle--
Skeletal muscle from
mdg pups or 1- to 2-day-old wild type mice were
obtained, and KCl-washed microsomes were prepared as described above.
The skeletal muscle calcium channel was enriched using a wheat germ
agglutinin column after solubilization as described above. The enriched
material was subjected to sucrose gradient fractionation as described,
and the fractions were analyzed by SDS-PAGE and immunoblotting.
Transient Transfection in tsA201 Cells and
Immunoprecipitation--
tsA201 cells were transfected using the
calcium phosphate method. Cells were lysed in buffer containing 50 mM Tris, 1% digitonin, 0.5 M NaCl, and
protease inhibitors, and the solubilized material was isolated by
centrifugation. The solubilized material was immunoprecipitated with
beads to which the antibody to the Tunicamycin Treatment--
TsA201 cells were transfected
as described above. Tunicamycin (10 µg/ml) was then added to the
medium, the cells were incubated for 2 days and immunoprecipitated as
described above.
Skeletal Muscle L-type Calcium Channel Complex Is Maintained in
To examine if the virally expressed
To examine the localization of
WGA enrichment of the channel complex indicates that, unlike
Similar to Chimeras of
To test this hypothesis, we took advantage of the ability of the
To examine incorporation of the chimeric
To assess if secondary interaction sites exist on the
To examine if the Role of the First Extracellular Loop of
The The 1 and auxiliary
2
,
, and
subunits. The
subunits are the least characterized,
and their subunit interactions are unclear. The physiological
importance of the neuronal
is emphasized by epileptic stargazer
mice that lack
2. In this study, we examined the
molecular basis of interaction between skeletal
1 and
the calcium channel. Our data show that the
11.1,
1a, and
2
subunits are still associated in
1 null mice. Reexpression of
1 and
2 showed that
1, but
not
2, incorporates into
1 null channels.
By using chimeric constructs, we demonstrate that the first half of the
1 subunit, including the first two transmembrane
domains, is important for subunit interaction. Interestingly, this
chimera also restores calcium conductance in
1 null
myotubes, indicating that the domain mediates both subunit interaction
and current modulation. To determine the subunit of the channel that
interacts with
1, we examined the channel in muscular
dysgenesis mice. Cosedimentation experiments showed that
1 and
2
are not associated. Moreover,
11.1 and
1 subunits form a complex in
transiently transfected cells, indicating direct interaction between
the
1 and
11.1 subunits. Our data demonstrate that the first half of
1 subunit is required
for association with the channel through
11.1. Because
subunit interactions are conserved, these studies have broad
implications for
heterogeneity, function and subunit association
with voltage-gated calcium channels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
11.1 subunit and auxiliary
2
,
1, and
1 subunits (6). This four-subunit composition of the channels is similar to that
of the neuronal voltage-gated channels (7, 8). The auxiliary
2
and
subunits enhance membrane trafficking of
the
11.1 subunit and modulate the
voltage-dependent kinetics of the channel (9). In addition
to the role of the
1 subunit in the trafficking of the
channel, it also has a crucial role in EC coupling as emphasized by the
absence of EC coupling and early lethality in mice that lack the
skeletal
1 subunit (10). The subunit interactions of the
2
and
subunits have been relatively well defined.
1 subunit is an auxiliary subunit with four
predicted transmembrane domains and intracellular N and C termini. The
role of the
subunit in the membrane trafficking of the channels and subunit interaction remain unclear. The
1 subunit was
the only
subunit that was originally known and is associated with
the skeletal muscle voltage-gated calcium channel (11, 12). However, the identification of a neuronal
2 subunit (13) renewed
interest in the
subunits and led to the identification of a number
of
subunits (14-16).
subunits of the voltage-gated calcium channels are known to be
capable of forming heterogenous complexes both in
vivo (17, 18) and in vitro. The
subunits and the
1 subunit possess highly conserved interaction regions
(19) allowing the formation of heterogeneous channel complexes. The
2
subunits modulate different
1
subunits in vitro (20), and presumably this heterogeneity extends to the in vivo situation. Such diversity of
interaction would potentially multiply the number of possible
combinations of channels with different biophysical and physiological
properties, which could then translate into fine modulation of a
variety of cellular responses.
subunits are relatively less well understood. Subunit interactions
of
subunits with the voltage-gated calcium channels are unknown,
and their ability to form heterogeneous complexes is unclear. To
address these questions, we took advantage of
1 null
mice. Mice that lack
1 have been generated by use of
conventional gene targeting strategy (21, 22). The mice have no
detectable phenotype, and we have demonstrated previously (21) that the subunits of the skeletal muscle calcium channel are expressed at wild
type levels in the
1 null mice. The lack of the
1 subunit also does not appear to diminish the ability
of the other subunits to assemble together and conduct voltage-gated
calcium currents. However, the absence of the
1 subunit
slows the inactivation kinetics and increases the amplitude (21, 22) of
L-type currents in the skeletal muscle. This led us to examine if the
L-type calcium channel is maintained as a complex in the
1 null mice. We hypothesized that if the channel were
still maintained as a complex in the
1 null mice, it
would serve as a valuable tool offering the potential to explore
subunit-subunit interactions in an in vivo environment.
1 null mice. To examine the ability of
the
subunits to form heterogeneous complexes, the
1
and
2 subunits were introduced into the skeletal muscle
of the
1 null mice via adenovirus-mediated expression,
and their ability to incorporate into the channel was examined. We
demonstrate that the
1 does incorporate, but the
2 does not. Consistent with these results,
1EGFP localizes to the t-tubules, whereas the
2EGFP does not demonstrate organized t-tubule
localization. Furthermore, by using a
1/
2
chimeric strategy, we demonstrate that the first half of the
1 subunit mediates its interaction with the calcium channel. Examination of the electrophysiological characteristics of the
chimeric subunits in
1 null myotubes showed that the
first half of the protein is sufficient to restore calcium conductance of L-type currents in
1 null myotubes.
1
subunit associates, we examined the channel in the muscular dysgenesis (mdg) mice. In the absence of
11.1 in the
mdg mice,
1 does not associate with
2
, indicating that
11.1 is necessary
for
1 to associate with the calcium channel. These
predictions are confirmed by coimmunoprecipitation of
11.1 and
1EGFP subunits from transiently transfected cells.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 null mice as described previously (23). The microsomes
were solubilized twice in a buffer containing 50 mM Tris,
1% digitonin, 0.5 M NaCl, and seven protease inhibitors
(benzamidine, phenylmethylsulfonyl fluoride, aprotinin, leupeptin,
calpain inhibitor, calpeptin, and pepstatin). The solubilized material
was subjected to wheat germ agglutinin (WGA) chromatography. The
material bound to the WGA column was eluted using
N-acetylglucosamine and concentrated. In some cases, the
material was used as the enriched voltage-gated calcium channel. For
purification, the WGA-eluted material was diluted and applied to a
diethylaminoethylcellulose column, and the bound material eluted with
buffer containing 50 mM Tris, 0.1% digitonin, and 100 mM NaCl.
1--
The rabbit
1 subunit
(GenBankTM accession number M32231) was subcloned into the
BamHI/BamHI site of the adenovirus shuttle vector. The resulting construct was checked for appropriate orientation by restriction analysis.
2--
The mouse
2 subunit
(GenBankTM accession number NM_007583) was excised from the
pCDNA3 vector using HindIII and BamHI and
subcloned into the HindIII/BamHI sites of the
adenovirus vector.
1EGFP and
2EGFP--
The
1EGFP and
2EGFP constructs were generated
by inserting the cDNA into the pEGFP-N1 vector
(Clontech) in-frame with the enhanced fluorescent
green protein (EGFP) using standard PCR techniques. The resulting
construct encoded the
subunit with the EGFP tag at the C terminus
of the protein. The resulting constructs were excised and subcloned
into the adenovirus vector.
1/
2 Chimera--
To generate this
construct, a fragment of the rabbit
1 cDNA was
amplified using a forward primer that includes a
HindIII site and the start codon (
1 forward,
5' CCC AAG CTT CCA CCA TGT CCC CGA CGG AAG CC 3') and the reverse
primer in the region after the second transmembrane domain (5' GTT GTG
GCG TGT CTT CCG CTT CTT CCT GAA GGC 3'). This reverse primer also
includes a stretch of residues homologous to the
2
subunit. Another PCR fragment was generated using the
2
cDNA as a template, with the forward primer (5' GCC TTC AGG AAG AAG
CGG AAG ACA ACG CCA CAA C 3') and a reverse primer that includes a stop
codon and a BamHI site (
2 reverse, 5' CGG GAT
CCC GTC ATA CGG GCG TGG TCC GGC G 3'). The products from the two
PCRs were mixed and reamplified using the
1 forward and
2 reverse primers. The resulting product was subcloned into the adenovirus shuttle vector into the HindIII and
BamHI sites. The final construct was sequenced to verify
integrity. This construct encodes a protein that includes the N
terminus to aa 133 of
1 and aa 129 through the C
terminus of the
2 subunit. This protein is detected by
an antibody to the
2 subunit.
2/
1 Chimera--
A
fragment of the
2 cDNA was amplified using the
forward primer that includes a HindIII site and the
start codon (
2 forward, 5' CCC AAG CTT CCA CCA TGG GGC
TGT TTG ATC GAG 3') and the reverse primer (5' CGG CCG CAG CAG GTA ATC
GTA GAA CTC GCT CGC CGC 3'). A fragment of the
1
cDNA was amplified using the forward primer (5' GCG AGC GAG TTC TAC
GAT TAC CTG CTG CGG CCG 3') and a reverse primer that includes a stop
codon and a BamHI site (
1 reverse, 5' CGT GGA
TCC CGT TAA TGC TCG GGT TCG GC 3'). The products from the two
PCRs were mixed and reamplified with the
2 forward and
1 reverse primers. The resulting product was subcloned
into the adenovirus shuttle vector into the HindIII and
BamHI sites. The final construct was sequenced to verify the
integrity. This construct encodes a protein that includes the N
terminus to aa 128 followed by aa 134 through the C terminus of the
1 subunit. This protein is detected by an antibody to
the
1 subunit.
1sspnEGFP Chimera--
This construct was
generated by standard PCR techniques to replace the first extracellular
loop of the
1EGFP with the first extracellular loop of
the unrelated four-transmembrane protein sarcospan (24). The first
extracellular loop of sarcospan includes the six amino acids RTDPFW.
The protein can be detected by an antibody to GFP.
11.1--
This construct has been described
previously (25).
1 Null Mice--
10 µl of primary particles or
diluted secondary particles (~1011 particles/ml) of the
adenovirus were injected into the tibialis anterior and the quadriceps
muscles of 2-5-day-old
1 null pups as described
previously (26). 3 to 5 weeks later, the mice were sacrificed and the
injected muscles collected. The tissue was processed for
immunohistochemistry or biochemical analysis as described below.
11.1 (26)), sheep 6 (
11.1 and
1a (27)), guinea pig 1 (
2
), guinea pig 11/15/16/77 (
1 (28)),
rabbit 239 (
2/
3 (13)), and AP83
(
-dystroglycan (29)).
-dystroglycan (AP83). To visualize the
11.1 subunit, the sections were labeled with the
monoclonal antibody to the
11.1 subunit (IIID5E1). To minimize the background, the sections were first treated with the Fab
fragment using the conditions described by the manufacturer (The
Jackson Laboratories). Cy3-conjugated secondary antibodies were used to
detect the primary antibodies. EGFP fluorescence was used to detect the
subunits. Immunolabeled sections were visualized with the ×60
objective using confocal microscopy (Bio-Rad). The bar
represents 10 µm.
1 null embryos. Myoblasts were isolated by enzymatic digestion with
0.125% (w/v) trypsin and 0.05% (w/v) pancreatin. After
centrifugation, mononucleated cells were resuspended in plating medium
containing 78% Dulbecco's modified Eagle's medium with low glucose,
10% horse serum, 10% fetal bovine serum, and 2% chick embryo
extract. Cells were then plated at a density of ~1 × 104 cells per dish on gelatin-coated plastic culture dishes
(BD Biosciences). Cultures were grown at 37 °C in 8%
CO2, and after the fusion of myoblasts (5-7 days), the
medium was replaced with fetal bovine serum-free medium, and
CO2 was decreased to 5%. In the case of cDNA
expression,
subunit cDNAs of interest and a separate plasmid encoding the T-cell membrane antigen CD8 were mixed at a 1:1 weight ratio and cotransfected with the polyamine LT-1 (Panvera, WI). CD8-transfected cells were identified by incubation with anti-CD8 antibody beads (Dynal, Norway).
(V
V1/2)/k)). Where
Amax is Gmax;
V1/2 is the potential at which A = Amax/2; and k is the slope factor.
The external solution in all cases was (in mM) 130 triethanolamine methanesulfonate, 10 CaCl2, 1 MgCl2, 0.001 tetrodotoxin (Sigma), and 10 HEPES titrated with triethanolamine(OH) to pH 7.4. The pipette solution was (in mM) 140 Cs+-aspartate, 5 MgCl2, 5 EGTA, 10 MOPS-CsOH, pH 7.2.
11.1 subunit (IIF7)
was coupled. The beads were then thoroughly washed in a buffer
containing 0.1% digitonin, 0.5 M NaCl, 50 mM
Tris, and two protease inhibitors (benzamidine and phenylmethylsulfonyl
fluoride). The bound material was eluted using SDS loading buffer and
immunoblotted. Aliquots of the cell lysate were also immunoblotted to
examine the expression of the proteins.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 Null Mice--
The generation of
1
null mice using conventional gene targeting strategy has been described
previously (21). The subunits of the calcium channel are expressed at
normal levels in these mice. This, in conjunction with the expression
of robust voltage-gated calcium currents in the skeletal muscle (21,
22), led us to examine if the calcium channel subunits are maintained
as a complex. The calcium channel was purified from the wild type and
1 null mice as described under "Materials and
Methods." The presence of all the subunits in the complex was
verified by Western blotting with antibodies specific to each subunit
of the channel (Fig. 1). The
1 subunit can be detected in the complex from the wild type mice but not in the
1 null mice. The
11.1,
2
, and
1a subunits can be detected in the purified material from both the wild
type and the
1 null mice, demonstrating that the
residual calcium channel is maintained as a complex in the
1 null mice.
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Fig. 1.
The skeletal muscle L-type calcium channel is
maintained as a complex in the 1
null mice. Purified calcium channels from the wild type
(WT) and
1 null mice were immunoblotted to
detect the presence of the subunits. The
11.1,
2, and
1a subunits are detected in both
the wild type and the
1 null mice, whereas
1 is only detected in the wild type mice, indicating
that the channel is maintained as a complex in
1 null
mice.
1 Can Be Stably Incorporated into the L-type Calcium
Channels of
1 Null Mice--
The
1
subunit was reintroduced into the skeletal muscle of the
1 null mice, via recombinant adenoviruses, to determine
whether it could be incorporated into the calcium channel of the
1 null mice. Expression of the protein was examined by
immunoblot analysis (Fig. 2A).
The muscle from the wild type mice showed expression of the
1 protein, whereas the
1 null mice did
not express any
1 protein. Robust expression of
1 recombinant protein was detected in the muscle of the
mice injected with the recombinant adenovirus, confirming that the
adenovirus-mediated expression may be successfully used for the
expression of this subunit in the skeletal muscle.
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Fig. 2.
Incorporation of an adenovirally
expressed 1 into skeletal L-type
calcium channel of
1 null
mice. A, adenovirally expressed
1 in
1 null skeletal muscle was detected by immunoblot
analysis. The blot was reprobed with an antibody to
2.
WT, wild type. B, calcium channels from
solubilized microsomes were enriched using WGA chromatography and
immunoblotted. The channel is enriched by WGA as indicated by the
presence of
2 in the WGA elution.
1 is
also enriched by the WGA, suggesting association with the calcium
channel. C, WGA-enriched material was subjected to linear
sucrose gradient fractionation and immunoblot analysis.
11.1,
2, and
1a subunits
comigrate on the sucrose gradient.
1 migrates in the
same fractions, confirming incorporation into the calcium channels of
the
1 null mice. D, muscle sections from
1EGFP injected muscle were double labeled with an
antibody to the sarcolemma protein,
-dystroglycan (
DG). E, muscle sections were double-labeled with an
antibody to
11.1.
1EGFP is localized to
the t-tubules and colocalizes with
11.1.
1 is incorporated
into the residual calcium channel complex of
1 null
mice, a WGA-enriched preparation containing highly enriched material
was examined for the presence of the
1.
1
is enriched by the WGA column, as is the rest of the channel as
indicted by the presence of the
2
subunit in the
elution. This suggests that
1 may be incorporated into
the channel complex (Fig. 2B). To confirm this further, the enriched material was subjected to sucrose gradient fractionation and
immunoblot analysis (Fig. 2C). Our results clearly
demonstrate that
1 cofractionates with the other
subunits of the calcium channel, indicating that indeed it is stably
incorporated into the calcium channel complex of
1 null
mice. A fraction of
1 is not associated with the
channel. It is possible that this pool represents free
1
that is not complexed with the channel due to overexpression of the
protein, because it is not observed in similar preparations from older
wild type skeletal muscle (data not shown).
1, a construct that
encodes
1 with an EGFP at the C terminus was engineered.
Expression of this protein in mammalian cells revealed that
1EGFP traffics to the plasma membrane, even in the
absence of the other subunits of the calcium channel (data not shown).
By using adenovirus-mediated expression, the EGFP tagged constructs
were introduced in the muscle of the
1 null mice.
Interestingly, the protein is also localized to the sarcolemma (Fig.
2D), as indicated by its colocalization with
-dystroglycan, a marker for the sarcolemma (30) (Fig. 2D). In addition,
1EGFP is localized to the
t-tubules (Fig. 2, D and E), as confirmed by its
colocalization with
11.1 (Fig. 2E).
2 Does Not Incorporate into the Skeletal L-type
Calcium Channels of
1 Null Mice--
The ability of
2 to incorporate into the calcium channel of
1 null mice was then examined using a similar approach.
The adenovirus encoding
2 allows robust expression of
the protein in the skeletal muscle of
1 null mice. There
is no detectable endogenous protein in the skeletal muscle of either
wild type or
1 null mice (Fig.
3A) as reported previously
(13).
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Fig. 3.
2 subunit is not
incorporated into the skeletal L-type calcium channel of
1 null mice. A,
adenovirally expressed
2 in
1 null
skeletal muscle was detected by immunoblot analysis. The blot was
reprobed with an antibody to
2. WT, wild
type. B, microsomes from the
1 null mice
expressing
2 were solubilized, and the channel was
enriched using WGA chromatography. The calcium channel is enriched by
the WGA as indicated by the presence of the
2 subunit in
the WGA elution. The
2 subunit is not enriched by the
WGA. C, solubilized microsomes were subjected to linear
sucrose gradient fractionation and immunoblot analysis.
11.1,
2, and
1a subunits
comigrate on the sucrose gradient.
2 subunit does not
comigrate with the calcium channel subunits, confirming that it is not
incorporated into the channel of
1 null mice.
D, muscle sections from
2EGFP-injected muscle
were double-labeled with an antibody to the sarcolemma protein,
-dystroglycan (
DG).
2EGFP localizes to
the sarcolemma. E, muscle sections were double-labeled with
an antibody to
11.1.
2EGFP is not
localized to the t-tubules and shows a punctate pattern.
2EGFP does not colocalize with
11.1.
1,
2 is in the void, indicating that that
2 may not be incorporated into the complex (Fig.
3B). To ascertain if the
2 subunit is weakly
associated with the complex and the steps leading to the WGA
chromatography disrupt this association, sucrose gradient fractionation
of solubilized microsomes from
2 injected mice was
examined for the presence of
2 and the other subunits of the channel (Fig. 3C). These results demonstrate that
2 does not comigrate in the same fractions as the rest
of the channel complex confirming that it is not incorporated into the
channel complex in
1 null mice.
1EGFP,
2EGFP traffics to the
plasma membrane in transiently transfected mammalian cells (data not
shown). Consistent with the biochemical data indicating the absence of
an association of
2 with the calcium channel, the
adenovirally expressed
2EGFP in the skeletal muscle of
the
1 null mice does not demonstrate an organized
t-tubule expression pattern (Fig. 3D). Interestingly, similar to that observed with the
1 subunit, the protein
is also localized to the sarcolemma (Fig. 3D) and
colocalizes with
-dystroglycan, a marker for the sarcolemma (Fig.
3E). In contrast to
1,
2
appears in punctate clusters within some muscle fibers (Fig.
3E). In other fibers, a more diffused pattern is observed
(data not shown). However, the protein is not localized to the
t-tubules as confirmed by the absence of colocalization with the
11.1 subunit (Fig. 3E).
1 and
2 Define
Interaction Domain of
1 with the Calcium
Channel--
The
subunits possess a similar four-transmembrane
domain structure with intracellular N and C termini. The first
extracellular loops of the
subunits have charged regions that are
conserved across the
subunits (12). We therefore hypothesized that
the first half of the
1 subunit, including the first two
transmembrane domains, might mediate the interaction of the protein
with the channel, placing the first loop in close proximity to the channel.
1 subunit, but not the
2 subunit, to
incorporate into the calcium channel. Chimeric proteins containing the
first two transmembrane domains of the
1 subunit and the
last two domains of the
2 subunit were generated (Fig.
4A). The ability of this
protein to incorporate into the channel was examined by techniques
similar to those described for the
1 and
2 subunits. The chimeric protein can be detected in
microsomes from the injected muscle (Fig. 4B).
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Fig. 4.
The first half of the
1 subunit interacts with the L-type
calcium channels. A, schematic of the
1/
2 chimera. The protein encodes the
first half of the
1 subunit, including the N terminus,
first extracellular loop, and first two transmembrane domains, fused to
the second half of the
2 subunit. B,
adenovirally expressed
1/
2 in
1 null skeletal muscle was detected by immunoblot
analysis. The blot was reprobed with an antibody to the
2 subunit. WT, wild type. C,
solubilized microsomes were subjected to sucrose gradient fractionation
and immunoblot analysis for subunits of the calcium channel. The
1/
2 comigrates with the subunits of the
calcium channel, indicating that it is incorporated. D,
schematic of the
2/
1 chimera. The protein
encoded includes the first half of the
2 subunit
followed by the second half of the
1 subunit.
E, adenovirally expressed
2/
1
in
1 null skeletal muscle was detected by immunoblot
analysis. The blot was reprobed with an antibody to the
2 subunit. F, microsomes from
1 null mice expressing
2/
1 were solubilized, and the channels
were enriched using WGA. The
2/
1 chimera
is in the void of the WGA, whereas the channel is in the WGA elution as
indicated by the presence of the
2, indicating that the
2/
1 chimera is not incorporated.
1/
2 protein into the calcium channel, the
microsomes from such a preparation were solubilized and subjected to
sucrose gradient fractionation and immunoblotting. Our data show that
the
1/
2 chimera comigrates in the same
fractions as the other subunits of the channel, confirming that it is
indeed incorporated into the channel complex (Fig. 4C).
Similar to that observed for the
1 studies described
above, in some experiments a pool of the
1/
2 chimera that is not associated with
the channel complex is observed (data not shown), presumably as a
result of overexpression.
1
subunit that allow interaction with the calcium channel, we generated a
construct that encodes the first two transmembrane domains of the
2 subunit and the last two transmembrane domains of the
1 (Fig. 4D). Similar to the other
adenovirally expressed proteins, the
2/
1
chimera showed robust expression in the skeletal muscle of the
1 null mice (Fig. 4E). The
2/
1 protein is not enriched with the
calcium channel as indicated by its presence in the void of the WGA
column, whereas the other subunits of the calcium channel are in the
WGA elution, as indicated by the presence of the
2
subunit (Fig. 4F). These results clearly demonstrate that
the
2/
1 chimeric protein does not
incorporate into the calcium channel of the
1 null mice.
1 and
1/
2
Chimera Restore Conductance of L-type Calcium Channels in
1 Null Myotubes--
We have demonstrated previously
(21) that the L-type voltage-gated calcium current conductance is
increased in
1 null myotubes. To examine the functional
effects of the
subunits and the chimeras, the different constructs
were introduced into the myotubes of the
1 null mice
(Fig. 5, A and B).
In agreement with the biochemical studies, re-expression of
1 and
1/
2 chimera reduced
the calcium conductance (142 ± 12.1 and 160.5 ± 16.7 pS/pF,
respectively) significantly compared with that of
1 null
myotubes (296.1 ± 10.8 pS/pF). Consistent with the lack of
incorporation of
2 and
2/
1, these chimeras did not alter the
conductance significantly (262.6 ± 20.4 and 276.4 ± 8.2 pS/pF, respectively). In all of the cases, no effect was observed on
the voltage dependence of current conductance (G-V curve),
the voltage of half-maximal conductance, V1/2, and
slope factor, k values (Table
I). These results demonstrate that the
first half of
1, which mediates subunit interaction, can
modulate the functional properties of skeletal L-type voltage-gated
calcium channels.
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Fig. 5.
Electrophysiological characterization of
the subunits in
1 null myotubes. A,
whole-cell calcium currents when
1 null or
1 null myotubes transfected with the indicated
constructs are depolarized to 0, +20, or +40 mV from a holding
potential of
40 mV. The pulse duration was 500 ms. B,
voltage dependence of calcium conductance for
1 null
myotubes or
1 null myotubes transfected with the
indicated constructs. The curves correspond to a Boltzmann fit of the
population mean with the parameters Gmax
(pS/pF), V1/2 (mV), and k (mV),
respectively, of 301.7, 19.8, and 6.1 for
1 null; 142.2, 21.4, and 5.8 for
1; 263.3, 16.1, and 5.9 for
2; 153.6, 20.5, and 4.8 for
1/
2 chimera; and 274.9, 18.2, and 5.9 for
2/
1.
Ca2+ conductance of the 1 null cells expressing
1,
2,
1/
2,
and
2/
1 subunits
1 Directly Interacts with
11.1--
To determine the subunit of the calcium
channel that interacts with the
1 subunit, we took
advantage of the muscular dysgenesis (mdg) mice. The
mdg mouse arose as a spontaneous mutation in the gene that
encodes the
11.1 (31) that results in the loss of any
detectable protein (32). In the absence of
11.1,
2
(32, 33) and
1a are still expressed.
We sought to examine
1 subunit in these mice and
determine whether, in the absence of
11.1,
1 is associated with
2
. The
WGA-enriched material from mdg and 1-2-day-old wild type
muscle microsomes was subjected to sucrose gradient fractionation and
immunoblot analysis. In the wild type mice, the
11.1,
2
, and a fraction of the
1 subunits
comigrate on the sucrose gradient (Fig.
6A), confirming that they are
associated in a complex. Interestingly, a large fraction of the
1 subunit does not appear to be in the complex and
migrates in the lower fractions, and presumably this is developmentally
regulated. In contrast, in the sucrose gradient fractions from the
mdg muscle, the
11.1 is not detected.
However, the
2
and the
1 subunits do
not comigrate (Fig. 6A), suggesting that the presence of the
11.1 subunit may be necessary for the
1
subunit to associate with the calcium channel.
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Fig. 6.
1
interacts with the
11.1. A, enriched
skeletal muscle calcium channels from muscular dysgenesis
(mdg) and 1-2-day-old wild type mice were subjected to
sucrose gradient fractionation and immunoblot analysis. In the wild
type mice,
2 and
1 comigrate, indicating
the presence of an intact channel complex. In the mdg mice,
1 and
2 do not comigrate, indicating that
11.1 may be required for
1 to be
associated with the channel. B, tsA201 cells were
transiently transfected with the indicated constructs. Cell lysates
were immunoprecipitated with an antibody to
11.1.
Aliquots of the lysate and the immunoprecipitated material were
examined for the presence of the
11.1 and the
1EGFP. The
11.1 immunoprecipitates with
the
1EGFP.
1 subunit directly interacts with the
11.1 subunit, subunit associations were examined in
transiently transfected mammalian cells. TsA201 cells were transiently
transfected with cDNAs encoding the
11.1 subunit,
the
11.1 subunit, and the
1EGFP or the
1EGFP subunit alone. Cell lysates were subjected to
immunoprecipitation using an antibody to the
11.1
subunit. Aliquots of the lysate and the immunoprecipitated material
were examined for the presence of the
11.1 and
1EGFP proteins (Fig. 6B). Our results show
that in the cells transfected with the
11.1 subunit, the
11.1 subunit is immunoprecipitated, but the EGFP antibody does not detect any endogenous protein. Coexpression of
11.1 and
1EGFP allows
coimmunoprecipitation, indicating complex formation. The
1EGFP is not immunoprecipitated in the absence of the
11.1 subunit, indicating specificity of interaction.
These results confirm that the
11.1 and the
1 directly associate in the absence of the other
subunits of the voltage-gated calcium channel.
1 in Subunit
Interaction--
Our studies demonstrate that the first half of the
1 subunit interacts with the calcium channel complex via
the
1 subunit. To examine the role of the first
extracellular loop of the
1 subunit in subunit
interaction, a chimeric subunit was generated. This chimeric subunit
(Fig. 7A) has the first
extracellular loop of the
1 subunit replaced by an
extracellular loop of the unrelated four-transmembrane protein
sarcospan (24) and EGFP at the C terminus. Transient transfection of
tsA201 cells with the
11.1 and
1sspn and
immunoprecipitation demonstrated that the two proteins associate (Fig.
7B). These results further support a role for the first two
transmembrane domains of the
1 subunit in subunit interaction.
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Fig. 7.
Role of the first extracellular loop in
subunit interaction. A, schematic of the
1sspnEGFP chimera. The first extracellular loop of
1 is replaced by an extracellular loop of the unrelated
protein sarcospan. B, tsA201 cells were transiently
transfected with the indicated constructs. Cell lysates were
immunoprecipitated with an antibody to
11.1. Aliquots of
lysate and immunoprecipitated material were examined for the presence
of the
11.1 and the
1sspnEGFP. The
11.1 immunoprecipitates with the
1sspnEGFP. C, cell lysates from cells
transfected with the
1EGFP constructs and either treated
with tunicamycin or untreated were immunoblotted with an antibody to
GFP. Tunicamycin treatment inhibited N-linked glycosylation
of
1EGFP. D, tsA201 cells were transiently
transfected with the indicated constructs and treated with tunicamycin.
Cell lysates were immunoprecipitated with an antibody to
11.1. Aliquots of the lysate and the immunoprecipitated
material were examined for the presence of the
11.1 and
the
1EGFP. The
11.1 immunoprecipitates
with the
1EGFP in the absence of N-linked
glycosylation.
1 subunit has an N-linked glycosylation
site in the first extracellular loop and is glycosylated in
vivo. To examine the role of this glycosylation in the interaction
of the
1 subunit with the
11.1 subunit,
cells transiently transfected with the
11.1 and
1EGFP constructs, as described above, were treated with
the aminoglycoside antibiotic tunicamycin, an inhibitor of N-linked glycosylation. Examination of cell lysates from
treated and untreated cells indicated that the
1EGFP
existed in a smaller molecular weight form in tunicamycin-treated
cells, indicating an absence of glycosylation (Fig. 7C).
Furthermore, the
1EGFP could be immunoprecipitated by an
antibody to the
11.1 protein, indicating that the lack
of glycosylation does not inhibit the association of the
11.1 subunit with the
1 subunit (Fig.
7D).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunits of the voltage-gated calcium channels are the
least characterized subunits of the calcium channels. In this study, we
have dissected subunit interactions and domains mediating subunit
interaction of the
1 subunit with the other components of the skeletal L-type voltage-gated calcium channel. Moreover, we
provide evidence for restricted subunit heterogeneity of the
subunits. The voltage-gated calcium channels have a similar structure
and subunit interactions, hence these studies have broad implications
for the interactions of the
subunits with the voltage-gated calcium
channels (Fig. 8).
View larger version (25K):
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Fig. 8.
Model of subunit interactions of the high
voltage-gated calcium channels. The subunit is depicted as a
four-transmembrane domain protein with predicted intracellular N and C
termini. The first half of the
subunit interacts with the
1 subunit. The first extracellular loop contains charged
residues and glycosylation sites. The interaction sites of the
2
(Gurnett et al. (9)) and the
subunit
(Beta Interaction Domain-Pragnell et al. (19)) with
the
1 subunit have been defined previously.
The subunit was originally identified in skeletal muscle (12). In
the recent past, a number of
subunits that are expressed in a
variety of tissues have been cloned (13-16), and their functional roles are only beginning to be revealed. In general, the
subunits appear to be inhibitory (7, 22, 30, 34). The important physiological
role for the
subunit is emphasized by the epileptic phenotype in
the stargazer mouse, a spontaneous mutant that lacks the
2 subunit (13).
It is well known that the 1 subunit modulates the
inactivation kinetics and the current amplitude of the skeletal muscle voltage-gated calcium channels (21, 22). The
1 subunit
is not, however, required for maintaining the integrity of the channel complex or its targeting to the t-tubules. This is in sharp contrast to
the distribution of the subunits in mdg mice and
1 null mice.
2
shows a punctate or
diffused pattern in mdg mice (33), whereas
11.1 is reduced or absent in
1 null mice
(10). Recent studies have indicated that, unlike
1 (10),
1 does not have a major role in membrane trafficking of
the
11.1 subunit as assessed by gating current
measurements (21) or in EC coupling (35). Our results clearly
demonstrate that the first half of the
1 subunit that
allows subunit interaction also allows the restoration of L-type
currents in
1 null muscle. Interestingly, despite the differences in L-type currents in the wild type and
1
null mice, we have not observed any gross morphological changes in the
muscle. Taken together, these data suggest that, at least in skeletal muscle,
1 is predominantly involved in modulating the
biophysical properties of the channel.
Subunit interactions are conserved across the subunits of the
voltage-gated calcium channels as indicated by the presence of highly
conserved interaction sites on the subunit (19) and in
vivo and in vitro subunit heterogeneity (17, 18). We have identified the interaction site of the
1 with the
calcium channel and predict that other
subunits interact with
1 subunits of the calcium channels similarly. Our
studies indicate that the
subunits may not be as capable of
functional heterogeneity as
subunits, thereby restricting the
number of possible subunit associations of the
subunits with the
voltage-gated calcium channels. However, the
subunits have
diverging homologies, with the
6 subunit being the
phylogenetically most closely related subunit to the
1
subunit (15, 16). Hence, it is possible that the
subunits might
possess the ability to form heterogeneous complexes, albeit to a
relatively limited extent as compared with the
subunits. This is
also suggested by cell expression studies indicating the ability of the
1 subunit to associate with the
11.2
subunit (36), although it is associated with the
11.1 in
native tissue. The
1 null mouse offers a good model
system to test the ability of the other
subunits to form
heterogeneous complexes in an in vivo environment.
The t-tubule is a specialized organized structure continuous with the
sarcolemma in the skeletal muscle. There is increasing evidence that
the targeting of proteins to these structures involves determinants
that are different from targeting to the sarcolemma (37). We have
demonstrated the localization of the 1 subunit in the
t-tubules by microscopy. To our knowledge, this is the first report of
localization of the
1 subunit in the skeletal muscle.
Interestingly, the
1 subunit is localized to both the sarcolemma and the t-tubules, unlike the other subunits of the calcium
channel, which are predominantly localized in the t-tubules (4).
Whether this is an effect of overexpression of the protein is unclear.
However, the localization of the protein at the plasma membrane in
transfected mammalian cells suggests that the
1 subunit contains the determinants for plasma membrane localization. In the
future, it would be interesting to determine whether the ability of the
1 subunit to localize to the t-tubules of the skeletal muscle, in addition to the plasma membrane/sarcolemma, is a result of
its association with the subunits of the calcium channel or whether the
protein has its own signals, like the
11.1 subunit (38),
to target it to the t-tubules, where it can then associate with the
calcium channel complex.
We demonstrate that the first half of the 1 mediates the
interaction of the subunit with the rest of the calcium channel. This
subunit interaction also allows the restoration of L-type calcium
conductance in
1 null myotubes. The absence of
N-linked glycosylation of the
1 subunit does
not prevent the protein from associating with the
11.1
subunit, suggesting that the N-linked glycosylation is not
the predominant mediator of the interaction between the
11.1 and
1 subunits. Interestingly, the
first extracellular loop of the
1 subunit is relatively
negatively charged (12) and has been suggested to be important in
mediating the biophysical properties of the
1 subunit
(22). We demonstrate that the first extracellular loop of the
1 subunit is not required for subunit interaction.
Because the first half of the
1 subunit interacts with
the calcium channel, presumably via the transmembrane domains, the
first extracellular loop would be predicted to be in close proximity to
the
11.1 subunit.
The subunit interactions of the subunits with the calcium channels
have been contradictory, with suggestions of a requirement (7) or lack
of requirement (36, 39) of the
2
subunit in the
association of the
subunit with the voltage-gated calcium channel.
The studies on the mdg mice indicate that the
2
and
1 subunits are not associated,
thereby precluding a direct interaction between the two subunits.
Interestingly, in the 1-2-day-old mice, there is a pool of
1 subunit that is not associated with the calcium
channel. The exact significance of this is unclear. It is possible that
during development, there is an excess of the protein generated, and
later
1 that is not incorporated into the calcium
channel is degraded. Alternatively, it is possible that during the
early stages of muscle development,
1 is associated with
other proteins in addition to the calcium channel and involved in other
unknown functions. Further studies are necessary to clarify the role of
the
1 subunits during early development. Our cell expression studies indicate that the
1 subunit directly
interacts with the
1 subunit. This is consistent with
studies that demonstrate a direct effect of the
6
subunit on the
13.1 protein in the absence of the other
known calcium channel subunits (40). It is therefore likely that the
requirement of the
2
subunit to mediate some of the
electrophysiological properties contributed by the
subunit reflect
conformational changes rather than direct subunit interaction.
These studies provide valuable insights into the subunit
interactions within the voltage-gated calcium channels. The past few
years have seen the discovery of a number of different
subunits and
the confirmation of the four-subunit composition of the voltage-gated calcium channels. Understanding the subunit interactions of the
subunits is an important step toward understanding the structural and
functional subunit interactions within the voltage-gated calcium channels.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank all the members of the Campbell lab for helpful discussions and critical reading of the manuscript and Melissa Hassebrock, Keith Garringer, Lindy McDonough, and Lindsay Williams for technical support. We thank the University of Iowa Diabetes and Endocrinology Research Center (supported by National Institutes of Health Grant DK25295) and the University of Iowa DNA Sequencing Core Facility. We also thank the University of Iowa Gene Transfer Vector Core, supported in part by the Carver Foundation and National Institutes of Health P30DK54759, for the generation of the adenoviruses.
![]() |
FOOTNOTES |
---|
* This work was supported by funding from the American Heart Association (to J. A. and C. A.), National Institutes of Health Grants RO1 AR 46448 (to R. C. and R. G.) and R01 HL 47053 (to R. C.), and the Muscular Dystrophy Association (to C. C. C. and V. A.).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.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 400 EMRB, Iowa City,
IA 52242. Tel.: 319-335-7867; Fax: 319-335-6957; E-mail:
kevin-campbell@uiowa.edu.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M208689200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EC, excitation-contraction; WGA, wheat germ agglutinin; GFP, green fluorescent protein; EGFP, enhanced fluorescent green protein; aa, amino acid.
![]() |
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