From the Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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In the present study, we investigated the role of
channel subunits in the membrane targeting of
voltage-dependent L-type calcium channel complexes. We
co-expressed the calcium channel pore-forming Voltage-dependent calcium channels are
heteromultimeric complexes composed minimally of one of the various
isoforms of the pore-forming Recently, we and others reported that native L-type calcium channels in
rabbit cardiac myocytes (class C L-type channels) are minimally
composed of an Little is known about the targeting signals that direct calcium
channels to the plasma membrane. We have recently demonstrated that the
rat Materials--
All reagents were obtained from general sources
unless otherwise noted. The calcium channel subunit-specific antibodies
used in this study including Card C, Generation of a Mutant Cell Culture and Transfection--
HEK-tsA201 cells were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) containing 10% fetal bovine serum (Life Technologies) and 1%
penicillin/streptomycin at 37 °C in 5% CO2. Transient
expression of different calcium channel subunits in HEK-tsA201 cells
was carried out using the calcium phosphate precipitation method
(4).
Immunofluorescence Staining and Confocal
Microscopy--
Transiently transfected HEK-tsA201 cells were stained
with calcium channel subunit-specific antibodies following the
procedures described previously (4, 19). Briefly, the transfected cells were fixed in precooled ( Immunoprecipitation and Immunoblotting--
Transfected tsA201
cells were homogenized, and crude membrane fractions were prepared as
described previously (4). For co-immunoprecipitation of
Intact Cell Radioligand Binding--
To detect membrane
localization of L-type calcium channels, ligand binding experiments
were performed using the dihydropyridine (DHP)1 radioligand
(+)-[3H]PN200-110 with intact cells transfected with
different combinations of the channel subunits. The experimental
procedures were as described previously (4). Scatchard analyses
were performed to assess Bmax and
Kd values.
Palmitoylation of the
Membrane particulate fractions from transfected cells expressing the
In confocal microscopy studies, cells expressing the The The Conserved Core Region of A Mutation of the BID Disrupted Channel Targeting and Subcellular
Localization of the An SH3 Motif Mutation of
To test whether lack of a membrane targeting function of
DHP Binding and Plasma Membrane Formation of L-type Calcium
Channels--
To confirm that the immunofluorescence staining of the
channel subunits observed along cell surfaces represented functional calcium channels at the plasma membrane, whole-cell DHP binding experiments were performed using the radiolabeled antagonist
(+)-[3H]PN200-110. Since (+)-[3H]PN
200-110 is a hydrophobic ligand, the specific binding obtained with
intact cells should reflect binding to calcium channels located on the
plasma membrane. Different combinations of the calcium channel
subunits, including wild-type
For cells transfected with wild-type
A Different Distribution Pattern of the
To test targeting of the channel subunits when all three subunits were
expressed, we co-expressed We previously demonstrated that co-expression of calcium channel
An interesting finding from our present study is that co-expressed
We have previously identified an SH3-like motif that is present in all
four In summary, we have demonstrated that the signals necessary for
membrane targeting of 1C
subunit with different accessory
subunits in HEK-tsA201 cells and
examined the subcellular localization of the channel subunits by
immunohistochemistry using confocal microscopy and whole-cell
radioligand binding studies. While the pore-forming
1C
subunit exhibited perinuclear staining when expressed alone, and
several of the wild-type and mutant
subunits also exhibited intracellular staining, co-expression of the
1C subunit
with either the wild-type
2a subunit, a
palmitoylation-deficient
2a(C3S/C4S) mutant or three
other nonpalmitoylated
isoforms (
1b,
3, and
4 subunits) resulted in the
redistribution of both the
1C and
subunits into
clusters along the cell surface. Furthermore, the redistribution of
calcium channel complexes to the plasma membrane was observed when
1C was co-expressed with an N- and C-terminal truncated
mutant
2a containing only the central conserved regions.
However, when the
1C subunit was co-expressed with an
1
interaction-deficient mutant,
2aBID
, we did not observe formation of the
channels at the plasma membrane. In addition, an Src homology 3 motif
mutant of
2a that was unable to interact with the
1C subunit also failed to target channel complexes to
the plasma membrane. Interestingly, co-expression of the pore-forming
1C subunit with the largely peripheral accessory
2
subunit was ineffective in recruiting
1C to the plasma membrane, while co-distribution of all
three subunits was observed when
2a was co-expressed
with the
1C and
2
subunits. Taken
together, our results suggested that the signal necessary for correct
plasma membrane targeting of the class C L-type calcium channel
complexes is generated as a result of a functional interaction between
the
1 and
subunits.
INTRODUCTION
Top
Abstract
Introduction
References
1 subunit together with
accessory
and
2
subunits (1, 2). The
subunits
are very hydrophilic proteins and are localized toward the cytoplasmic
face of the plasma membrane (1, 2). Four different
subunit genes
have been cloned so far (3). All four different
subunits contain
two conserved domains in the central region flanked by unique N and C
termini (3). In contrast to the cytoplasmic orientation of the
subunit, the
2
subunit consists of the extracellular
2 peptide disulfide-bonded to the membrane-spanning
-peptide (1, 2). It has been shown that the accessory subunits play
important roles in modulating calcium channel function (1).
Co-expression of a
subunit with an
1 subunit in
heterologous expression systems results in an increase in the number of
binding sites for drugs or toxins known to bind to the channels, an
increase in peak current amplitude, and an increase in the number of
channels at the cell surface (4-8). The increase in peak current
amplitude observed upon
subunit co-expression is probably due to
the increase in the number of plasma membrane-localized channels
(5-7). The effects of the
2
subunit and the
molecular events underlying
2
-mediated regulation of
the channels are less well understood (1). Co-expression of the
2
subunit with the cardiac
1C subunit
resulted in increased peak current amplitude and altered channel
kinetics in Xenopus oocytes (9, 10). However, kinetic
changes were not observed in other studies (11, 12).
1C subunit, a
2 subunit,
and an
2
subunit (13, 14). Co-localization of the
calcium channel
and
2
subunits with the
1 subunits in the T-tubule membranes of heart cells was
observed (13). Direct interactions between
1 and
subunits (15, 16) or between
1 and
2
subunits (17) have been identified, and the regions responsible for
subunit interaction have been partially mapped out in each subunit
(15-18). However, more biochemical studies are required to further
understand the modulatory effects of the
2
and
subunits.
2a isoform is palmitoylated, while other
isoforms, including the
1b,
3, and
4 subunits, are not (5). The palmitoylation sites have
been mapped to Cys3 and Cys4 in the N terminus
of the rat
2a subunit (5). Palmitoylation appears to
play a role in the targeting of the
2 subunit to the plasma membrane when it is expressed in the absence of an
1 subunit (19). Palmitoylation-deficient
2a mutants and other nonpalmitoylated
isoforms were
localized intracellularly when expressed in the absence of other
channel subunits (19). In addition, palmitoylation appears to confer
unique regulatory properties to the rat
2a subunit (20).
However, it remains unclear whether palmitoylation is an important
signal for targeting of channel complexes. Here, we further
investigated the role of
subunits in plasma membrane targeting of
calcium channel complexes. To identify the region in
subunits
required for targeting, we have expressed the
1C subunit
with different wild-type and mutant
subunits in HEK-tsA201 cells
and assessed the localization of channel subunits using confocal
microscopy and whole-cell radioligand binding studies. Since the
membrane and extracellular orientation of the
2
subunit might suggest a role in targeting other channel subunits to the membrane, the effect of the
2
subunit in membrane
targeting of the channel complexes was also studied.
EXPERIMENTAL PROCEDURES
GEN, and
anti-
2 antibodies were described previously (4, 5). The
expression vector used with the
2
subunit,
pMT21
2
, was as described (13). Generation and
expression of several mutant
subunits including
2aCys
,
2aIFP, and
2aBID
were described previously (19). The
monoclonal anti-
2 antibody was a generous gift from Drs.
Sylvie Vandaele and Michel Lazdunski (21).
Subunit Expression Construct--
To
construct a truncation mutant of the
2a subunit,
2a-(17-411) (
2a
NC), the
5'-untranslated region and initiator ATG were amplified by the
polymerase chain reaction using the following oligonucleotide primers:
5'-AAGCTTAGCAACAGCTCGGTCAGG-3' (plus strand) and
5'-GGATCCCATGAAGAGGTGGCAGGACG-3' (minus strand). This initial fragment
was cloned as a HindIII-BamHI fragment into a modified version of pCR3 (Invitrogen) containing a C-terminal KT3 epitope tag (4) in frame with a BamHI site.
Subsequently, a fragment encoding amino acids 17-411 of
2a was amplified by polymerase chain reaction using the
following primers: 5'-GGATCCGCAGACTCCTACACCAGC-3' (plus strand)
and 5'-AGATCTGTGGGTGGCCTTCCAGTACGC-3' (minus strand). The
2a-(17-411) fragment was cloned into the
BamHI site of the previously described vector containing the
2a initiator ATG and KT3 epitope tag as a
BamHI-BglII fragment; orientation was confirmed by restriction endonuclease analysis.
20 °C) methanol/acetone (1:1) for 5-10 min at 4 °C and followed by incubating with labeling buffer (1% bovine serum albumin in phosphate-buffered saline, pH 7.4) to block
nonspecific binding. Different primary antibodies were diluted in
labeling buffer and incubated with cells for 1-2 h at room temperature. The secondary antibodies including fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG, Alexa 488-conjugated
goat anti-rabbit IgG, and tetramethylrhodamine
isothiocyanate-conjugated rabbit anti-goat IgG (Molecular Probes, Inc.,
Eugene, OR) were used subsequently. The cellular distributions of the
expressed channel proteins were visualized using a Zeiss LSM-10 laser
scanning microscope.
1 and
subunits, membrane particulate fractions were
solubilized in solubilization buffer (50 mM Tris, pH 7.4, 5 mM EDTA, 5 mM EGTA, 0.4 M NaCl, 1%
Triton X-100, and 0.1% SDS containing protease inhibitors (4)).
Solubilized proteins were immunoprecipitated overnight with agitation
at 4 °C using
GEN antibodies coupled to protein
G-Ultralink resin (Pierce). Immunoprecipitates were washed with
solubilization buffer 3-5 times and eluted with SDS sample buffer.
Immunoblotting procedures were performed as described previously (4).
Detection of immunoreactive bands was performed using either enhanced
chemiluminescence (Pierce) or colorimetric enhanced diaminobenzidine
substrate reaction (Pierce).
RESULTS
2a Subunit Is Not Necessary
for Functional Channel Formation--
Previously, we had demonstrated
that co-expression of the rat
2a subunit with the
1C subunit resulted in an increase in the number of
functional channels at the cell surface as assessed by
immunohistochemical and electrophysiological analyses (4). We and
others subsequently confirmed this finding by electrophysiological measurements of whole-cell charge movement (5-7). Since the
2a subunit is a palmitoylated protein (5), it was
possible that palmitoylation was playing a role in targeting channels
to the plasma membrane. However, co-expression of either the wild-type
2a subunit or the palmitoylation-deficient
2a(C3S/C4S) subunit with the
1C subunit
resulted in an increase in the amount of measured whole-cell
charge-movement, suggesting that palmitoylation was not required for
the formation of channel complexes (5). In order to obtain further
insight into the role of palmitoylation of the
2 subunit
in the formation and membrane targeting of
1C complexes,
transiently transfected cells expressing the
1C subunit alone or the
1C subunit in combination with either the
wild-type
2a or the mutant
2a(C3S/C4S)
subunits were analyzed biochemically as well as by confocal
immunofluorescent microscopy.
1C subunit in combination with either the
2a or the
2a(C3S/C5S) subunit were
solubilized as described under "Experimental Procedures." Following
immunoprecipitation of solubilized proteins with the
GEN
antiserum, immunoblotting was used to detect the presence of the
1C and
2a proteins in the
immunoprecipitated pellets. Immunostaining of the immunoprecipitates
with the Card C antiserum revealed that the
1C subunit
co-purified with both the
2a and
2a(C3S/C4S) subunits (Fig.
1A, top),
suggesting that the loss of palmitoylation in the
2a(C3S/C4S) mutant did not noticeably disrupt
interactions between the
1C and
2a
subunits. The presence of the
2a and
2a(C3S/C4S) subunits in the immunoprecipitated pellets
was confirmed by immunoblotting with the
2a antiserum (Fig. 1A, bottom).
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Fig. 1.
Palmitoylation of the subunit was not necessary for the targeting of functional
channels to the cell surface upon
subunit
co-expression. Transiently transfected cells expressing the
1C subunit alone or in combination with either the
wild-type
2a subunit or the palmitoylation-deficient
2a(C3S/C4S) mutant were analyzed by both
co-immunoprecipitation and immunohistochemical studies. A,
solubilized membrane fractions from transfected cells were
immunoprecipitated with the
GEN antiserum.
Immunoblotting with the
2a antiserum (bottom)
was used to confirm the immunoprecipitation of
2a
protein. Immunoblotting with the Card C antiserum (top) was
used to detect the amount of
1C protein, which was
co-immunoprecipitated, presumably through interaction with the
2a subunit. B represents the typical Card C
staining pattern observed in transiently transfected cells expressing
the
1C alone. No discernible immunostaining is seen at
the cell surface. C, staining of
1C
2a cells with the Card C antibody
(left) revealed punctate
1C distribution at
and/or near the plasma membrane, similar to previously published
results (4). Similarly, the staining pattern of the
2a
antibody revealed a punctate distribution of the
2a
subunit in these co-transfected cells. This confocal image section
was taken from near the top surface of the cell. D,
staining of
1C
2a(C3S/C4S) cells with
the Card C antibody (left) also revealed punctate
1C distribution along the cell surface (top view).
Additionally, immunostaining with the
2a antibody
revealed a punctate localization of the
2a(C3S/C4S)
protein at the plasma membrane as well.
1C
subunit alone exhibited a perinuclear staining pattern (Fig.
1B, left; the phase image is shown on the
right), while co-expression of the
1C subunit
with the
2a subunit resulted in a striking change of the
1C subunit staining to a punctate pattern at the plasma
membrane (Fig. 1C, left). Both of these findings
are consistent with previous results (4). Interestingly, when
co-expressed with the
1C subunit, the staining pattern
of the
2a subunit also changed to a more punctate and
less continuous pattern (Fig. 1C, right) along
the cell surface compared with the continuous membrane staining of
cells expressing the
2a subunit alone that we observed
previously (19). It was of interest to determine if a similar or
different pattern of staining would be observed when the
1C subunit was co-expressed with the
palmitoylation-deficient
2a(C3S/C4S) subunit.
Co-expression of the
1C subunit with the
2a(C3S/C4S) mutant resulted in punctate staining of the
1C subunit at the cell surface (Fig. 1D,
left panel). Notably, co-expression with the
1C subunit also led to redistribution of the
2a(C3S/C4S) protein to the plasma membrane (Fig.
2D, right), despite
the fact that this mutant protein, when expressed in the absence of the
1C subunit, was localized intracellularly (19). These
results support the hypothesis that the multimerization of channel
subunits, which likely occurs during or shortly after synthesis of the
individual peptides, allows the proper folding and assembly of channel
complexes and facilitates their transport to the cell surface. From
these results, as well as previously reported electrophysiological
results (5), the lack of palmitoylation of the
2a
subunit did not appear to affect subunit interactions that are critical
for channel formation and targeting.
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Fig. 2.
Redistribution of calcium channel
1C subunits by
co-expression with different
subunits.
The
1C and different
subunits were co-expressed in
tsA cells. The cells transiently transfected with
1C
1b (A),
1C
3 (B), or
1C
4 (C) were
immunofluorescently stained with the Card C antibody to reveal the
expression patterns of the
1C subunits, and
visualization was with confocal microscopy. A phase-contrast image of
1C
4-transfected cells was included as an
example to show the plasma membrane outlines of the cells
(C). Punctate channel clusters were observed along cell
membranes in all three types of transfected cells, and the distribution
patterns were similar to those seen in Fig. 1, C and
D.
1C Subunits Were Redistributed to the Plasma
Membranes by Co-expression with Different
Isoforms--
To test
whether the three other
isoforms can form complexes with and cause
membrane targeting of the
1C subunit, we co-expressed the
1C subunit with the
1b,
3, or
4 subunits in tsA201 cells. The
subcellular localization pattern of the
1C subunit was
revealed by immunostaining with the Card C antibody. Punctate staining of
1C subunit clusters was observed along plasma
membranes in
1C
1b-,
1C
3-, and
1C
4-transfected cells (Fig. 2). A
phase-contrast image of
1C
4-transfected
cells was used as an example to confirm the plasma membrane outlines of
the cells (Fig. 2C, right). Although the
1b,
3, and
4 subunits were
nonpalmitoylated and were intracellularly localized when expressed
alone (5, 19), all three
subunits were able to cause redistribution
of the
1C subunit from a perinuclear location to the
plasma membrane. These results suggested that each
subunit was able
to form complexes with the
1C subunit in a manner
similar to that observed with the
2a
palmitoylation-deficient mutant, confirming that the palmitoylation of
the
subunits was not required for membrane targeting of calcium
channel complexes when co-expressed with the
1C subunit.
Subunits Is Sufficient for
Membrane Targeting of Channel Complexes--
The finding that each
subunit caused a similar redistribution of the
1C
subunit to the plasma membrane suggested that a structural domain
common to each
subunit might be important. All four different
subunits discovered to date (including numerous splicing variants)
contain a conserved central region with two highly homologous domains
(3). Within this region, a site that mediates interaction with various
1 subunits has been identified and termed the
-interaction domain (BID) (16, 22). However, it has not been
demonstrated whether the conserved core region of the
2
subunit is sufficient for membrane targeting of the
1C
2 complex. To test this possibility, an
N- and C-terminal truncation mutant of the rat
2a
subunit,
2a
NC, containing only the conserved core
region, was created and transfected into HEK-tsA cells. An
intracellular staining pattern of this minimal
subunit was observed
when this truncation mutant was expressed alone (data not shown). This
finding was consistent with the fact that the N-terminal palmitoylation
sites (membrane-anchoring sites) were deleted from this truncation
mutant (19). To study the membrane targeting function of
2a
NC, the
1C and
2a
NC subunits were co-expressed in tsA cells, and the
expression pattern of the
1C subunit was revealed using
the Card C antibody. Clusters of
1C subunits were
observed as bright punctate spots on the cell surface, as shown in the
confocal image generated from a top section of the cell (Fig.
3, top panel). A
confocal image taken from the middle section of the cell showed
punctate formation of channel complexes along the cell plasma membranes
(Fig. 3, top panel). These staining patterns were
similar to those seen in Figs. 1 and 2. The ability of the
2a
NC subunit to allow targeting of the
1C
2 complex to the cell plasma membranes
indicated that the conserved core region of
subunits was sufficient
for the targeting of the
1C·
complexes. Taken
together, these results suggested that the membrane targeting observed
in the presence of all
subunits can be achieved with only the
conserved region. However, the unique electrophysiological properties
of calcium channels that have been observed upon co-expression of
different
isoforms were probably determined by distinctive N- and
C-terminal regions (1, 20).
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Fig. 3.
The conserved core region of the
subunits is required and sufficient for membrane
targeting of channel complexes. The
2a
NC
subunit, a truncation mutant of
2a, was co-expressed
with the
1C subunit in tsA cells. The expression pattern
of the
1C subunits was revealed by the Card C antibody
using confocal microscopy. The confocal image shown in the
top panel was obtained from the top surface of
the cell, and the channel clusters were observed as bright round spots
on the cell surface. The image shown in the lower
panel was obtained from a middle section across the cell,
and the staining pattern was punctate along the cell plasma membrane.
The staining patterns of the
1C subunit shown here are
similar to those observed in Figs. 1 and 2.
2a Subunit--
The results
presented so far suggested that the signal for membrane targeting of
the
1C and
subunits generated upon formation of
1C·
complexes, since membrane targeting was
achieved in most instances with subunits that exhibited an
intracellular distribution when expressed alone. If this is true, then
mutations that disrupt subunit interaction should disrupt membrane
targeting. Electrophysiological studies in Xenopus oocytes,
as well as studies with fusion proteins, have suggested that the BID in
the conserved domains of
subunits is critical for
subunit
interaction (16). Mutation of Pro237 to Arg in the BID of
the
1b subunit appeared to completely eliminate
1
interaction (16). However, studies with full-length
and full-length
1C subunits have not been performed
to determine if this BID region was also critical for channel targeting
to the plasma membrane. Therefore, site-directed mutagenesis was used
to create a mutation at Pro234 (analogous to
Pro237 of the
1b subunit) in the
2a subunit. The wild-type
2a subunit or
the
2a(P234R) mutant was transiently expressed in tsA201
cells in combination with the
1C subunit and assessed
for subunit interactions. Membrane particulate fractions from these
cells were solubilized as described under "Experimental
Procedures," and the soluble fractions were subsequently
immunoprecipitated with the
GEN antibody. Immunoblotting
with Card C and
2a antisera was used to detect the
presence of the
1C and
2a subunits,
respectively, in the immunoprecipitated pellets. Immunoprecipitation of
the wild-type
2a subunit from
1C cells
resulted in co-purification of the
1C subunit in the
immunoprecipitated pellet (Fig.
4A, top
left). By contrast, immunoprecipitation of the
2a(P234R) protein did not result in co-purification of
the
1C subunit. The
1C subunit was seen
in both the input and the flow-through lanes from
1C
2a(P234R) cells (Fig. 4A,
right), indicating that the absence of
co-immunoprecipitation was not due to lack of
1C protein
but rather to a lack of interaction between the
1 and
proteins. Transiently transfected
1C
2a(P234R) cells were fixed and
immunohistochemically stained with the Card C and
2a
antibodies (Fig. 4B). The
1C protein was
localized to perinuclear regions in these cells (Fig. 4B,
left), similar to the distribution observed in cells
expressing the
1C subunit alone (see Fig.
1B). Similarly, the
2a(P234R) subunit
exhibited a diffuse intracellular staining pattern (Fig. 4B,
right) indistinguishable from that of
2a(P234R) expressed alone (19). Thus, disruption of the
interaction between the
1 and
subunits caused by the mutation in BID also disrupted membrane targeting of channels in these
cells. Alternatively, mutation of Pro234 may have affected
subcellular localization and channel targeting through global
disruption of the
2a protein structure.
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Fig. 4.
Mutation of the BID prevented channel
targeting and altered the subcellular localization of the
2a protein. A,
transiently transfected cells expressing the
1C subunit
in combination with either the wild-type
2a subunit or
the
2a(P234R) mutant were used to assess the effect of
this mutation on the ability of the
1C and
2a subunits to co-purify as a complex. Solubilized
fractions from transfected cells were immunoprecipitated with the
GEN antiserum. Subunits were detected by immunoblotting
with the Card C (upper left) and
2a (lower left) antibodies. The
input and flow-through fractions (6% of each) from
1C
2a(P234R) cells were immunoblotted with
the Card C antiserum (right), indicating that the lack of
1C protein in the
GEN immunoprecipitate
was not due to the absence of
1C protein. B,
transiently transfected
1C
2a(P234R) cells
were immunohistochemically stained with the Card C and
2a antibodies and analyzed by confocal
immunofluorescence microscopy. Card C staining in these cells was
perinuclear (left), similar to the distribution pattern
observed in cells expressing the
1C subunit alone (Fig.
1B). Interestingly, the staining by the
2a
antibody revealed a diffuse intracellular distribution of the
2a(P234R) protein (right).
2a Disrupted Channel
Targeting and Interaction with the
1C Subunit--
An
SH3-like motif was found in the conserved region of all four
subunits (19). We have reported that a triple point mutation in this
SH3 motif (
2a(I115A/F117A/P119L), or
2aIFP) of the
2a subunit resulted in a
marked reduction of palmitoylation and an intracellular rather than
membrane localization of the protein (19). The mutated amino acids
correspond to residues previously identified to play key roles in the
SH3 domain of Src (23, 24). Here, we investigated the effect of the SH3
domain on membrane targeting of the channel complexes. The SH3 motif
mutant,
2aIFP, and the
1C subunits were
co-expressed in tsA cells, and expression patterns of both the
1C and mutant
2aIFP subunits were
visualized using confocal microscopy. Interestingly, co-expression of
2aIFP with
1C did not result in
redistribution of the channels. As shown in Fig.
5A, both the
1C
(left) and
2aIFP (right) subunits remained in cytoplasmic regions. The staining patterns of both subunits
were indistinguishable from those observed with either subunit
expressed alone in cells (19).
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Fig. 5.
An SH3-motif mutant disrupted the interaction
between the 1C
and
2a subunits
and failed to target the channels to the plasma membrane. A,
the
1C and an SH3 mutant of
2a,
2aIFP, were co-transfected into tsA cells. The
expression patterns of both the
1C and mutant
2a subunits were revealed by immunofluorescent staining
with Card C (left) or the
2 antibody
(right), respectively. The confocal images showed
intracellular staining patterns for both subunits. B, the
crude membrane fractions were prepared from
1C
2a- or
1C
2aIFP-co-transfected cells, and
solubilized proteins were immunoprecipitated with the
GEN antibody. The immunoprecipitates were analyzed with
SDS-polyacrylamide gel electrophoresis and immunoblotting. The
wild-type
2a and
2aIFP mutant subunits
immunoprecipitated by
GEN were detected with the
2 antibody on the immunoblot (bottom).
However, the
1C subunit was only co-immunoprecipitated
with the wild-type
2a subunits (top,
lane 1, detected with the Card C antibody), but
not with the
2aIFP mutant subunits (top,
lane 2). Lane 3 shows the
presence of the
1C subunits in the flow-through fraction
from immunoprecipitation of the
1C
2aIFP
cells.
2aIFP is due to disruption of
1
interaction, association between
1C and
2aIFP was analyzed using immunoprecipitation. Membrane particulate fractions were prepared from
1C
2aIFP-co-transfected cells, and
solubilized proteins were immunoprecipitated with the
GEN antibody. As a positive control,
co-immunoprecipitation of the
1C and wild-type
2a subunits was easily detected (Fig. 5B, lane 1). However, the
1C subunit
did not co-immunoprecipitate with the
2aIFP (Fig.
5B, lane 2) subunits, suggesting that
the interaction between these two subunits was largely impaired. The
1C subunit in
1C
2aIFP-co-transfected cells was detected
in the supernatant fraction of the
GEN
immunoprecipitation (Fig. 5B, lane 3),
indicating that lack of
1C
2aIFP
co-precipitation was not due to lack of
1C expression.
Taken together, these results suggested that the SH3 domain of the
subunits may play a role in
1
interaction.
Alternatively, the mutations may have disrupted post-translational
modifications or proper folding of the
subunit and caused a loss in
normal function, although the mutant
subunit was able to be
recognized by the
2 and
GEN antibodies,
suggesting a lack of global disruption of protein structure.
1C
2a,
1C
2a(C3S/C4S),
1C
2a
NC,
1C
2aIFP, and
1C
2aBID
, were transfected
into tsA cells. Whole-cell binding experiments were performed, and
results were subjected to Scatchard analysis.
1C
2a,
1C
2a(C3S/C4S), or
1C
2a
NC, saturable DHP binding was
observed in all three cases. The Bmax and
Kd values were between 0.5-1.2 pmol/mg protein and
0.2-0.6 nM, respectively, from four independent
experiments, and no appreciable differences among these subunit
combinations were observed (Table I). In
contrast, no saturable binding was obtained from cells transfected with
either
1C
2aIFP or
1C
2aBID
. These DHP binding
results were in agreement with the expression patterns observed in the
immunofluorescence staining studies, suggesting that the functional
L-type calcium channels were targeted to the cell plasma membrane in
1C
2a-,
1C
2a(C3S/C4S)-, and
1C
2a
NC-transfected cells. However,
when plasma membrane expression of channel complexes was not observed
in the immunostaining studies, as for the cases of
1C
2aIFP and
1C
2aBID
, no specific DHP
binding was obtained as well.
Whole-cell DHP binding in tsA cells transfected with calcium
channel subunits
1C
2aBID
and
1C
2aIFP subunits, Kd values
were not determined (ND). No significant differences were observed in
the binding parameters between the cells transfected with different
channel subunits as indicated by statistical analyses. The values are
expressed as mean ± S.E.
1C Subunit
Was Obtained upon Co-expression with the
2
Subunit--
Recently, it has been demonstrated that
1
subunits can associate with
2
subunits through direct
interactions between the transmembrane domains of
1 and
the
peptide and the extracellular regions of
1 and
the
2 peptide (17, 18). To address the possibility that
the
2
subunit also can play a role in the membrane targeting of calcium channel complexes, we transiently expressed the
1C and
2
subunits in tsA cells. Card C
and a monoclonal anti-
2 antibody were used to reveal the
expression patterns of the
1C and
2
subunits, respectively. When the
2
subunit was expressed alone in the cells, the majority of the protein localized to
the plasma membrane and exhibited smooth rather than punctate staining
(Fig. 6A). We also observed
some faint intracellular staining of
2
, which may
reflect newly synthesized or partially processed proteins. This finding
is different from that previously obtained by Brice et al.
(25), who reported an intracellular staining pattern of
2
subunits when expressed in COS-7 cells. This
inconsistency may be explained by different glycosylation and
processing machinery in specific cells, since the highly glycosylated
2
subunit may require proper processing and
glycosylation to be inserted into the plasma membrane (26).
Co-expression of the
1C and
2
subunits
in tsA cells did not allow significant targeting of the
1C subunit to the plasma membrane (Fig. 6B). Only a very small portion of the
1C subunits localized
to the cell membrane (Fig. 6B, indicated by
arrows), while the majority of the
1C
subunits remained intracellular (Fig. 6B). This result suggested that the
2
subunit was unable to mimic the
targeting function observed with the
subunits. In addition, since
the
2
subunit itself did localize to the plasma
membrane, the results suggest that complex formation between
1C and
2
is less efficient than
between
1C and
subunits. This suggestion is
consistent with the fact that the
2
subunit-mediated
regulation of channel function is less dramatic than that observed with
the
subunits (6, 9, 10).
View larger version (33K):
[in a new window]
Fig. 6.
Co-expression of
2
with
1C subunits did not redistribute
the
1C subunits into clusters
along the cell surface. A and B are confocal
images showing expression patterns of the
2
and
1C subunits. A, the cells transfected with
the
2
subunits alone were stained with a monoclonal
anti-
2
antibody. A plasma membrane expression pattern
was observed. B, the
1C and
2
subunits were co-transfected into tsA cells, and
the cells were stained with Card C to visualize the expression pattern
of the
1C subunits. A faint plasma membrane staining was
obtained (indicated by a right arrow); however,
the majority of the
1C subunits remained intracellular.
C, the cells co-transfected with
1C
2
subunits together, and stained
with Card C,
2, and
2 antibodies to
reveal the expression pattern of each subunit. Similar punctate plasma
membrane staining patterns were observed for all three subunits.
1C,
2a, and
2
subunits together into tsA cells. All three
subunits localized to the plasma membrane as shown in Fig.
6C, and similar punctate staining patterns along the plasma
membrane were observed for all three subunits. Interestingly, the
punctate staining pattern for the
2
subunit in
1
2
-co-transfected cells was
different from the smooth staining seen in the cells transfected with
the
2
subunit alone (Fig. 6, compare C
(right) with A). The punctate pattern of the
2
subunit when co-expressed with other subunits is
consistent with channel complex formation. In addition, saturable DHP
binding was obtained in
1C
2
-co-transfected cells,
confirming that functional channels were targeted to the plasma
membrane (data not shown). Moreover, when the
1C
2
-co-transfected cells were co-stained with antibodies for the
1C and
2
subunits or the
2a and
2
subunits, overlays of confocal images indicated
that all three subunits were co-localized along cell plasma membranes (data not shown). These results further demonstrated the importance of
the
subunits in membrane targeting, since co-expression of the
subunit was necessary for the proper formation of channel complexes
composed of all three channel subunits (compare Fig. 1C with
Fig. 6C).
DISCUSSION
subunits with
1 subunits resulted in formation of
clusters of channels along the cell plasma membranes (4). In the
present study, we further investigated the structural determinants of the
subunits for membrane targeting. Although palmitoylation was
required for membrane localization of the rat
2a subunit when this subunit was expressed in the absence of other subunits in tsA
cells (19), the results presented here clearly demonstrated that the
palmitoylation state of the
subunits was not a major determining
factor for plasma membrane targeting of channel complexes when
subunits were co-expressed with the
1C subunit. We
examined the expression patterns of channel complexes in
1C
2a-,
1C
2aCys
-,
1C
1b-,
1C
3-, and
1C
4-co-transfected cells, and punctate clusters of channels were observed at the plasma membrane in all cases
despite the fact that only the wild-type
2a was
palmitoylated. In addition, we demonstrated that the central conserved
core region of the
subunits was necessary and sufficient for
membrane targeting of
1C·
complexes. Thus, the
N-terminal variable regions containing the palmitoylation sites in the
rat
2a subunit were not required for the targeting and
punctate distribution of channel complexes. However, several mutations
in the conserved region of the
2a subunit that disrupted
1
interactions resulted in a failure to target the
1C and
subunits to the plasma membrane. Taken together, our results suggested that a functional interaction between
the
1C and the conserved domains of the
subunits is required for membrane targeting of the class C L-type channels. Interestingly, our results demonstrated that the targeting we observed
also was responsible for movement of the
subunits to the plasma
membrane. For example, although the
2a(C3S/C4S) and the
2a
NC mutants were localized intracellularly when
expressed alone (19), upon complex formation with the
1C
subunit, both of these mutant subunits redistributed to the plasma
membrane, and exhibited a punctate staining pattern similar to the
1C subunit. Since neither
1C nor these
mutant
subunits, nor the wild-type
1b,
3, and
4 subunits, are able to target to
the plasma membrane when expressed alone, these results strongly
suggest that the signals for membrane targeting are contained in the
complex of
1C·
subunits rather than in either
subunit alone. Thus, it is not surprising that mutations that disrupt
complex formation, such as the
2aBID
and
2aIFP mutants, disrupt membrane targeting. Conceivably, a membrane targeting "element" is formed upon the association of
the
1C and
subunits. This membrane targeting element
may involve a region in either one of the subunits that is exposed due
to subunit association or may involve an intersubunit domain.
1C
complexes formed punctate clusters along cell
plasma membranes. Channel clustering has been identified for several channel types. For example, shaker K channels and NMDA receptors are
found to form clusters along cell membranes through association with
PDZ-domain containing proteins (27, 28). Our results suggested that
either the
1C or the
subunits, or alternately, complexes of
1C and
subunits, may form secondary
interactions with other cellular proteins such as PDZ-domain-containing
proteins, and this secondary protein-protein interaction may result in
channel clustering at specific plasma membrane locations (hot spots). However, the mechanisms underlying calcium channel clustering are not
clear at this point. Of interest, the association between the
1C and
2
subunits did not appear to be
sufficient or strong enough to target the
1C subunit to
the plasma membrane, although
1
2
interactions have been described (17, 18). Alternatively, the
association of
1 and
2
may require the
presence of the
subunit, since the conformation assumed by the
1·
complex may allow better recruitment of
2
to the complex. When
1
2
subunits were co-expressed in
cells, channel clusters containing all three subunits were observed
along plasma membranes. Our present findings may also explain previous
electrophysiological results in mammalian cells that demonstrated that
the effect of the
2
subunit in augmenting calcium
channel currents was much less significant compared with that of the
subunits, while the effects of
and
2
were
synergistic (12).
subunits known to date (19). SH3 domains have been shown to
mediate protein-protein interaction in many important signaling
processes (29). The SH3 motif of the
subunits is in the conserved
core region and about 100 amino acids upstream of the BID domain (19).
There are two possibilities that may account for our finding that
mutations in the SH3 motif of
subunits disrupted interaction with
the
1C subunit. First, since the SH3 motif is adjacent
to the BID region in the
subunits, mutations in the SH3 domain may
interfere with direct binding between
1 and the BID
domain of
subunits. Alternatively, the SH3 motif may be a
structurally important determinant of the
subunits, and mutations
in this region may disrupt the important protein-protein interactions
or important structural domains in the
subunits. Our previous
findings that the palmitoylation level of
2aIFP was
significantly decreased also suggested the structural importance of the
SH3 motif (19). Key residues in the SH3 domain may play important roles
in maintaining proper conformation of
proteins.
1C and
subunits are likely to
be generated by the formation of the complex of
1C and
subunits. While the
1C subunit, as well as several
mutant and wild-type
subunits were unable to target to the plasma
membrane when expressed alone, complexes of these subunits were
targeted to the plasma membrane, where they exhibited a punctate
distribution. In contrast, the
2
and
1C subunits were unable to achieve this targeting in the
absence of the
subunits. The results pointed to a key role of
subunits in membrane targeting, and the region in the
subunit that
was responsible for targeting was narrowed to the two domains conserved
in all
subunits. Moreover, an SH3 motif in the
subunits has
been suggested to play a role in membrane targeting and association
with the
1 subunits. Our studies provided novel
biochemical evidence for better understanding the structural determinants of accessory subunit-mediated regulation of L-type calcium channels.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL23306 (to M. M. H.).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.
Recipient of NIMH, National Institutes of Health, National
Research Service Award Predoctoral Fellowship MH10770.
§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., S215, Chicago, IL 60611. Tel.: 312-503-3692; Fax: 312-503-5349; E-mail: mhosey{at}nwu.edu.
The abbreviations used are:
DHP, dihydropyridine; BID, -interaction domain; SH3, Src homology 3.
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REFERENCES |
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