From the Department of Biochemical Pharmacology, University of
Innsbruck, A-6020 Innsbruck, Austria
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INTRODUCTION |
Voltage-activated Ca2+ channels are expressed in a
multitude of tissues and are involved in a variety of cell functions
(1). The skeletal muscle L-type Ca2+ channel,
also called dihydropyridine
(DHP)1 receptor, is localized
in the junctions between the transverse tubules and the sarcoplasmic
reticulum (2, 3), where it primarily functions as the voltage sensor in
excitation-contraction coupling (4, 5). It consists of five subunits:
the pore-forming
1S subunit, the
2
subunit complex, a peripheral membrane protein, the
1a
subunit, and the
subunit, which is unique for this tissue (6-8).
Besides functioning as the ion channel proper, the
1S subunit contains the binding sites for specific drugs (9, 10) and
domains for interactions with the Ca2+ release channel (11)
and the accessory channel subunits (12). The molecular domain
responsible for binding the
subunit is a conserved motif of nine
amino acids in the cytoplasmic loop between repeats I and II of the
1 subunit. Mutations within this motif severely
interfered with
binding to
1 and with
-induced current stimulation (13).
The
subunit exists in several isoforms and splice variants in
different tissues (14). Coexpression of various combinations of
1 subunits and accessory subunits in heterologous
expression systems has contributed much of our present knowledge about
the functions of the subunits. Whereas the
1 subunit
alone is sufficient for the expression of functional Ca2+
channels (15, 16), additional expression of
2
and
subunits increases the incorporation of functional channels into the
plasma membrane and modulates the current properties (17-19).
coexpression has been demonstrated to enhance the Ca2+ and
DHP binding capacity of
1C (20), to elevate current
magnitudes, and increase gating charges (21). A multitude of
qualitative effects of
on Ca2+ currents has been
reported. However, they are as variable as the subunit combinations and
expression systems used for these studies (14, 22). The most compelling
evidence for functional modulation of current properties by
are the
increase in open probability in single-channel recordings of
1C co-transfected with
2a in
Xenopus oocytes (23, 24) and in mammalian cells, a
modulation of current densities but not of gating charges upon coexpression of
1C with a mutated
2a
subunit (25). As possible mechanisms for modulatory effects of
, an
improved intramolecular coupling (26) and a stabilization of
Ca2+ coordination sites in the channel pore have been
suggested (20).
In several recent studies, the mechanism by which
increases channel
expression in the plasma membrane has been directly addressed. In
transiently transfected HEK cells, it was shown with
immunocytochemistry that the cardiac
2a subunit is
preferentially localized in the plasma membrane and that coexpression
of the cardiac
1C with
2a causes the
translocation of
1C into the membrane (25). A similar
translocation of a neuronal
1A subunit into the plasma
membrane was accomplished by four different
isoforms:
1b,
2a,
3b, and
4 (27). Interestingly, not all of the
subunits that
are able to induce
1A translocation were localized in
the plasma membrane. Only
1b and
2a but
not
3b and
4 showed a plasma membrane
association. Palmitoylation at two N-terminal-located cysteines is a
possible mechanism by which
2a could be anchored to the
plasma membrane. However, mutation of these cysteines that blocked
palmitoylation of
2a expressed in tsA201 cells caused a
failure of current stimulation but not of channel incorporation as seen
in recordings of gating charges (28).
The skeletal muscle L-type Ca2+ channel is
distinct from other voltage-gated Ca2+ channels with
respect to its organization in the triad junction (29), with respect to
its function in excitation-contraction coupling (4), and with respect
to modulation of its channel properties (14, 30). Whether interactions
between the skeletal muscle
1 and
subunits expressed
in mammalian non-muscle cells also differ from those of other isoforms
is not known. Here we report isoform-specific effects of
1a and
2a on the subcellular distribution
and current stimulation upon coexpression with
1S in
tsA201 cells. A mutation in the
interaction domain of
1S interferes with the stable association of the
subunits but not with the
1a-induced stimulation of
Ca2+ currents.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
tsA201 cells, a HEK cell subclone stably
transfected with the SV40 large T-antigen, were plated and proliferated
in F-12 medium (Life Technologies, Inc.) containing 10% fetal bovine
serum. Cells were grown to 80% confluency before passing. For
structural analysis (green fluorescent protein (GFP) and
immunocytochemistry), cells were plated at dilutions of about 1:10 onto
poly-L-lysine-coated 13-mm round coverglasses and
transfected on the following day (see below) when cells reached 30%
confluency. For patch clamp analysis, cells were plated in 35-mm
culture dishes at a dilution of 1:10. After transfection at about 50%
confluency, cells were replated onto poly-L-lysine-coated
25-mm round coverglasses at dilutions between 1:5 and 1:10 to get
isolated cells for patch clamp recordings on the following day.
Transfection--
Transfections were carried out with a
liposomal transfection reagent (DOTAP, Boehringer Mannheim) according
to the manufacturer's instructions. The total amount of DNA used per
35-mm culture dish was 10 µg, 5-8 µg of which was specific DNA
(expression plasmids encoding
1S constructs,
, and
GFP); the rest was filled up with inert DNA (pUC18; Ref. 31). In
co-transfection experiments, two or more expression plasmids were
combined at equimolar concentrations. This resulted in coexpression of
GFP with any
1S construct in approximately 70% of
GFP-transfected cells. The liposome/DNA mixture was diluted in 1.5 ml of F-12 medium and then added to the culture. On the following day,
the cells were processed for immunocytochemistry or replated for patch
clamp analysis.
Expression Plasmids (see Table
I)--
The coding sequence of the
rabbit
1S-cDNA was excised from the plasmid pCAC6
(33) by HindIII digestion and inserted into the expression
plasmid pcDNA3 (Invitrogen, San Diego, CA).
Mutations were introduced into
1S by site-directed
mutagenesis of the
1S-pcDNA3 using the splicing by
overlap extension technique (32). In a first step, a SacII
(nt 81 of
1S)-XhoI (nt 2652 of
1S) fragment of the
1S-cDNA was
subcloned from the
1S-pcDNA3 into the pBluescript
SK(
) (Stratagene, La Jolla, CA). In a second step, the mutations in
the
1S were generated by using different mutagenic
primers: 1, the sense primer
5'-AAGCAGCAGCTAGAGGAGGACCTTCGGGGCTCCATGAGCTGGAT-3' for a single
substitution in the
1S in position 366 from tyrosine to
serine (
1S-Y366S); 2, the sense primer
5'-GCCAAGTCCAGGGGAACCTTCCTGAGAGAAGGAAAGCTG-3' for a 30-amino acid
deletion mutation in the
1S from position 351 to
position 380 (
1S-
351-380); and 3, the sense primer
5'-GCCAAGTCCAGGGGAACCTTCTGGATCACGCAGGGCGAG-3' for an 18-amino acid
deletion mutation in the
1S from position 351 to
position 368 (
1S-
351-368). To facilitate the
identification of positive clones, a PvuII site (nt 1071 of
1S) was eliminated by a silent mutation in the mutagenic
primers. The mutated
1S fragments were inserted into the
1S-pcDNA3 after digestion with SacII
and XhoI. All mutations were verified by sequence
analysis.
The construction of a
1a-GFP C-terminal fusion protein
required five steps. In the first step the GFP-cDNA was excised
from the GFP-pRK5 by using a BamHI (nt 2 of GFP) and a
HindIII site (nt 1671 of GFP-pRK5) and ligated into the
pBluescript SK(
). Secondly, the SacI (nt 695 of
1a)-BamHI (multiple cloning site of pSVL)
fragment of the rabbit
1a-cDNA from pSVL (20) was inserted into the GFP-pBluescript SK(
). In the third step, a fragment
consisting of the 3' terminus of the
1a-cDNA fused
to the 5' terminus of the GFP-cDNA including a
BamHI restriction site was constructed by PCR using the
fusion primer: 5'-CTCATGGGATCCATCATGGCGTGCTCCTGCTGTTGGGGCACC-3'. In the fourth step, NarI (nt 1147 of
1a) and BamHI (nt 2 of GFP) digestion of the
polymerase chain reaction product allowed insertion into the
pBluescript SK(
) subclone containing the
1a- and
GFP-cDNAs. Finally, the fused
1a-GFP fragment was
inserted into the
1a-pcDNA3 after digestion with
BstXI (nt 834 of
1a) and ApaI
(multiple cloning site of pcDNA3). The integrity of the
1a-GFP transition was verified by cDNA sequence
analysis.
GFP and Immunofluorescence Labeling--
Paraformaldehyde-fixed
cultures were immuno-stained as described previously (37). For
double-labeling with GFP, Texas red-conjugated antibodies (Jackson
Immuno Research, West Grove, PA) were used to exclude bleed-through
between the red and the green channels. Working dilutions and the
sources of primary antibodies are listed in Table
II. Samples were evaluated on a Zeiss
Axiovert microscope with epifluorescence and phase contrast optics and
documented on 35-mm high speed black and white film. The antibodies
were carefully characterized for their use in immunofluorescence
experiments in previous studies (37-39). Controls, like the omission
of primary antibodies and incubation with inappropriate antibodies,
were routinely performed.
Patch Clamp Recording--
Electrophysiological recordings were
performed using the whole-cell configuration of the patch clamp
technique (42). Cultures grown on 25-mm-round coverglasses were mounted
in a recording chamber and viewed with a 16× phase contrast
multi-immersion lens on a Zeiss Axiovert microscope. Fluorescent cells
(
GFP or GFP-transfected) were selected for recording, except for
controls with nontransfected cells. The bath solution contained (40 mM BaCl2, 100 mM tetraethylammonium chloride, 10 mM HEPES, and 5 µM (±)-BayK8644
(adjusted to pH 7.4 using tetrathylammonium hydroxide). Patch pipettes
pulled from borosilicate glass and fire-polished were filled with 130 mM cesium aspartate, 10 mM HEPES, 2 mM MgATP, 2 mM CsEGTA, and 0.5 mM
MgCl2 (adjusted to pH 7.4 with CsOH). Resistances of the
patch pipettes were between 4 and 7 megaohms. An Axopatch 200A patch
clamp amplifier controlled by the software pClamp 6.0 (Axon
Instruments, Foster City, CA) was used for all recordings. Capacitative
currents were compensated using the built-in analog circuits (series
resistance error was corrected for 80%). Leak resistance in the
cell-attached mode was normally larger (8 gigaohms). Current data were
low-pass Bessel-filtered with 1 kHz and sampled at 0.5 kHz with an
IBM-compatible PC. To test cells for the expression of barium currents,
a voltage ramp protocol was used (from
80 mV to +70 mV over a period
of 750 ms) without using any leak current subtraction. Under these conditions, the current detection threshold, indicated by an inward peak in the current trace (see, e.g. Fig. 6a),
was about 10 pA. Nontransfected tsA201 cells or cells transfected only
with
GFP showed one or, in rare cases, two distinct types of inward
barium currents. One with the current peak at
11.5 mV ± 2.1 mV
S.D. during the voltage ramp and with fast inactivation kinetics
characteristic for T-type current; a second current peaking at 8.7 mV ± 5.1 mV S.D. (see also Ref. 43). To exclude a possible
interference with the analysis of the heterologously expressed
currents, any current peaking below +30 mV was rejected from the
analysis. Only cells transfected with the
1S constructs
showed inward barium currents, with the current peak located between
+30 mV and + 50 mV of the voltage ramp. These high voltage-activated
currents were therefore attributed to the expression of one of the
1S constructs. For a further check, I/V curves were
determined for those cells showing stable recordings with a high
current density using a voltage step protocol with a holding potential
of
80 mV and 1 s test pulses of
40 mV in 10 mV increments to
+80 mV (using a
P/4 leak subtraction protocol). The I/V curves and
current kinetics (activation time constant
act = 18.2 ms ± 3.5 ms S.E., inactivation time constant
act = 486 ms ± 41 ms S.E. at +40 mV test potential and
1S +
GFP coexpression; see Fig. 6b) showed similar characteristics when
1S and
1a
were coexpressed in L-cells (18) or in myotubes (44).
 |
RESULTS |
1a and
1S Are Expressed in
Different Cellular Compartments--
An expression plasmid encoding a
fusion protein of the
1a subunit of the skeletal muscle
DHP receptor and GFP, called
GFP, has been constructed as a tool for
studying the subcellular distribution of
1a transiently
expressed in tsA201 cells.
GFP aids in the identification of cells
expressing the
1a subunit and allows its direct
localization. Double-labeling of transfected tsA201 cells with GFP and
immunofluorescence using an antibody against the
subunit resulted
in identical distribution patterns of both stains (Fig.
1, a and b).
Transfection with the
GFP fusion protein (Fig. 1b) or
transfection with the wild type
1a (Fig. 1e)
also resulted in identical
immuno-labeling patterns. Thus,
GFP
fluorescence gives a true picture of the
1a
localization, and using the fusion protein instead of the wild type
1a, has no undesired effects on its subcellular
localization. This was still true when the
distribution was altered
upon coexpression with the
1S subunit (see below).
Moreover, electrophysiological analysis showed that effects of
GFP
and wild type
1a on Ca2+ currents were
indistinguishable in control experiments in which the cardiac
1C was coexpressed with
GFP or
1a2 and upon coexpression of both
constructs with
1S in dysgenic skeletal myotubes
(53). Thus, our
GFP construct can be
used in place of
1a, with the advantage of allowing the
direct observation of
1a expression and localization in
transiently transfected cells. The distribution pattern of
GFP when
expressed by itself in tsA201 cells was always diffuse and evenly
distributed throughout the cytoplasm (Fig. 1a). The label
did not appear to be specifically associated with any membrane system
or cytoplasmic structure. Only the nuclei and vacuolar structures
excluded the
GFP stain. This was different from the fluorescence
signal obtained by expressing GFP alone, which was also diffusely
distributed throughout the cytoplasm but in addition labeled the nuclei
(Fig. 1d).

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Fig. 1.
Subcellular localization of the DHP receptor
1a subunit expressed in tsA201 cells. A fusion
protein of 1a and GFP ( GFP) is expressed in a tsA201
cell (a-c) and localized with GFP fluorescence
(a) and by immunofluorescence with a antibody
(b). Both localization methods give a very similar diffuse
distribution pattern of GFP. Cells transfected with GFP alone also
show a diffuse fluorescence, but unlike with GFP, the nuclei are
brightly labeled (d). tsA201 cells transfected with the wild
type 1a and immuno-labeled with anti- (e)
show the same diffuse distribution as GFP (cf. b).
e and f, phase contrast (Ph) images of
cells shown in a, b and in e,
respectively. N, nuclei of transfected cells;
bar, 10 µm.
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The
1S subunit of the DHP receptor expressed in tsA201
cells showed a different subcellular distribution. Localizing
1S with a specific antibody showed that it was contained
in a dense network of a tubular membrane compartment that extended
throughout the entire cytoplasm (Fig. 2,
a, c, and e). The tubular/reticular network was very dense in the perinuclear region, and occasionally the
nuclear envelope was also labeled. The reticular nature of the
compartment could be best seen in thin regions of the cells. Based on
these structural characteristics, the
1S-containing compartment is identified as the ER. Other cytoplasmic compartments that could have been recognized by their morphology, most importantly, the plasma membrane, were not labeled with the
1S
antibody. Transfection of tsA201 cells with
1S
constructs in which the
interaction domain has either been deleted
or mutated showed the same subcellular distribution in the ER (see Fig.
2, a, c, and e).

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Fig. 2.
Subcellular localization of the wild type and
two mutants of the DHP receptor 1S subunit. tsA201
cells transfected with 1S (a and
b), 1S-Y366S (c and
d), and 1S- 351-380 (e and
f) were immunofluorescence-labeled with an antibody against
the 1S subunit. All three 1S constructs
are expressed in a cytoplasmic membrane system that is densest in the
perinuclear region of the cells (saturated fluorescence signal around
the nuclei (N)). Focusing on thin regions of the cells
reveals the reticular nature of the 1-containing
membrane system. a, c, and e are
enlargements of the regions indicated by boxes in the
corresponding phase contrast (Ph) images (b,
d, and f). Bars, 10 µm.
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Coexpression of
1a and
1S Causes the
Redistribution of
1a but Not of
1S--
GFP expressed alone was diffusely
distributed throughout the cytoplasm (Fig.
3, a and b).
However, when
GFP was coexpressed with the wild type
1S subunit of the DHP receptor, the
1a
subunit became localized in the tubular/reticular membrane system,
presumably the ER (Fig. 3, c and d). The
cytoplasm was essentially free of diffuse
GFP fluorescence, and no
staining of the plasma membrane could be observed. Double-labeling of
GFP and an antibody against
1S showed a
colocalization of
1a and
1S in the ER
(see Fig. 4, a and
b). To test whether this redistribution of
GFP was due to
its direct association with the
1S subunit, three
1S constructs with mutations in the
interaction
domain have been generated. In
1S-
351-380, the
entire
interaction domain has been deleted; in
1S-
351-368, the N-terminal portion of the
interaction domain has been deleted; in
1S-Y366S,
tyrosine in position 366 has been substituted with serine. Equivalent
mutations in the
interaction domain of
1A have been
shown to inhibit binding of
1b and current stimulation
(12). If the translocation of
GFP from the cytoplasm to the ER was a
result of a direct association of
1a to
1S, mutations in the
interaction domain of
1S should inhibit the observed translocation of
GFP.

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Fig. 3.
Subcellular distribution of 1a
coexpressed with the wild type and mutant 1S
subunits. tsA201 cells were transfected with GFP alone
(a and b) or in combination with
1S (c and d),
1S-Y366S (e and f), and
1S- 351-380 (g and h).
1a is localized by GFP fluorescence (upper
row) and with immunofluorescence using an antibody against the subunit (lower row). When coexpressed with the wild type
1S, the 1a subunit is localized in a
reticular cytoplasmic membrane system (arrow; c
and d). Upon coexpression with 1S constructs
mutated in the interaction domain, 1a remains
diffusely distributed throughout the cytoplasm (e-h),
similar to the distribution pattern in cells in which GFP has been
expressed alone (a and b). N, nuclei;
bar, 20 µm.
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Fig. 4.
Comparison of the subcellular localization of
1a and 1S in tsA201 cells coexpressing
GFP with the wild type and mutant 1S subunits.
1a localization is shown with GFP fluorescence
(upper row), and 1S localization is shown
with immunofluorescence (lower row). GFP and wild type
1S are colocalized in a reticular cytoplasmic membrane
system (a and b). 1S-Y366S
(d) and 1S- 351-380 (f) are
both localized in the reticular membrane system; however, in these
cells GFP is not associated with the reticular membrane system but
diffusely distributed in the cytoplasm (c and e).
Insets show a small field of a thin area of the cell at
2-fold magnification. N, nuclei; bar, 10 µm.
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Indeed, when
GFP was coexpressed with the
1S
constructs in which the
interaction domain had been mutated, the
GFP distribution was diffuse (Fig. 3, e-h) and
indistinguishable from that of
GFP expressed alone (Fig. 3,
a and b). Quantitative analysis of the
GFP
distribution when expressed alone or in combination with either one of
the
1S mutants showed that 376 of 441 (85.3%) analyzed cells co-transfected with
1S had a clearly identifiable
ER distribution of
GFP, but in none of the cells co-transfected with
1S-Y366S or
1S-
351-380 was
GFP
found in the ER (Table III). The small fraction of cells in
GFP/
1S co-transfected cultures
that did not show an ER distribution of
GFP most likely represents
cells that only expressed
GFP and not
1S and cells
with an overall poor morphology. The differential distribution of
GFP upon coexpression with the different
1S
constructs was verified by immunolocalization of
1a
(Fig. 3, b, d, f, and h).
Immuno-labeling of
1a or
GFP gave the same
distribution patterns as seen with intrinsic
GFP fluorescence (Fig.
3, a, c, e, and g). 348 of
447 (77.9%) analyzed cells co-transfected with
GFP and
1S, but none of the cells co-transfected with either one
of the
1S mutants showed
immuno-localized in the ER.
Since the two deletion mutants,
1S-
351-368 and
1S-
351-380, gave identical results in all
experiments, quantitative data and figures of only
1S-
351-380 will be shown in this article as representative results for both constructs. Coexpression of the
1S constructs with the wild type
1a gave
the same results in that only cells co-transfected with
1a and the wild type
1S showed an ER
distribution pattern of
immunostain (see Table III). Thus, the
coexpression of
GFP or
1a with the wild type
1S caused the redistribution of the
1a
subunit from the cytoplasm to a tubular/reticular membrane compartment,
presumably the ER, but this redistribution fails when the
interaction domain in the
1S subunit is mutated.
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Table III
Subcellular distribution of 1S, 1a, and GFP in
transfected tsA201 cells
Values are given in cell numbers and percentage for each individual
construct; balance to 100% represents cells with labeling patterns
that could not unambiguously be identified as ER or cytoplasmic (Cyt.)
stain.
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Double-labeling of the
1a subunit and the
1S constructs with GFP and a specific antibody,
respectively, showed that upon coexpression of
GFP and the wild type
1S,
GFP was colocalized with the
1S
subunit (Fig. 4, a and b). The ER labeling
patterns of
1S and
GFP were identical. However, when
GFP was expressed together with
1S-Y366S or
1S-
351-380,
GFP remained diffuse and was not
colocalized with the tubular/reticular compartment containing the
mutant
1S subunits (Fig. 4, c-f). In
contrast, no changes in the distribution of the wild type or mutant
1S constructs occurred upon coexpression with
GFP.
This indicates that specific interactions between the
interaction
domain of
1S and
1a cause the association
of
GFP with the
1S subunit at the ER. However,
coexpression of
1S and
1a does not result in a recognizable change of
1S distribution within the
cell.
Coexpression of
2a and
1S Causes the
Redistribution of
1S but Not of
2a--
Expression of the cardiac isoform of the DHP
receptor
subunit (
2a) alone or in combination with
the skeletal muscle
1S subunit gave a different
distribution pattern than that seen with
1a. tsA201
cells transfected with
2a and immuno-stained with the
antibody showed an unequivocal plasma membrane stain (Fig. 5a). The periphery of the
cells was outlined by a continuous fine line, and the cytoplasm was
essentially free of immuno-label. The plasma membrane stain was more
clearly seen in regions where the plane of the membrane was parallel to
the optical axes rather than in flat parts of the cells, where the
membrane was almost perpendicular to the optical axes. This plasma
membrane label of
2a did not change when it was
coexpressed with any of our
1S subunit constructs (Fig.
5, c, e, and g). However, the distribution pattern of wild type
1S changed upon coexpression with
2a. Whereas the ER and nuclear envelope label could
still be seen in some cells, most tsA201 cells coexpressing
2a and
1S showed a distribution of
1S in the periphery of the cells (Fig. 5d).
279 of 395 (70.6%) cells co-transfected with
2a and
1S had a clearly identifiable peripheral localization of
1S (Table IV). The
remaining cells, which for the most part had
1S
localized in the ER, could often be identified as those that expressed
1S without
2a. The peripheral
1S stain was colocalized with the
2a
immuno stain in the plasma membrane. However, the
1S
label was not continuous throughout the plasma membrane but occurred in
intensively labeled aggregates in or near the plasma membrane. These
aggregates were of variable sizes and shapes. The
1S
distribution was always distinct from that of the
2a.
Whereas
2a was sometimes more intensive in the
1S aggregates, more often it was not, and it always
continued between the
1S aggregates. Thus,
1S is colocalized with
2a, but they do
not coexist throughout the plane of the membrane.

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Fig. 5.
Comparison of the subcellular localization of
2a and 1S in tsA201 cells coexpressed
with the wild type and mutant 1S subunits. tsA201
cells were transfected with the cardiac 2a alone
(a and b) or in combination with
1S (c and d),
1S-Y366S (e and f), and
1S- 351-380 (g and h) and were
double-immunofluorescence-labeled. 2a (upper
row) is localized in the periphery of the cell
(arrows). This label is best seen in areas where the plasma
membrane is oriented parallel to the optical axes. Wild type
1S is localized in discrete aggregates in the cell
periphery (d, examples indicated by open
arrowheads) and in the nuclear envelope (filled
arrowheads). 1S-Y366S (f) and
1S- 351-380 (h) do not form these
peripheral clusters but remain localized in a tubular/reticular
membrane system, presumably the ER (double arrowheads).
N, nuclei; bar, 10 µm.
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Table IV
Subcellular distribution of 1S, and 2a in
transfected tsA201 cells
Values are given in cell numbers and percentage for each individual
construct; balance to 100% represents cells with labeling patterns
that could not unambiguously be identified as plasma membrane (PM) or
ER stain.
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This translocation of
1S from the ER to the plasma
membrane upon coexpression with
2a was only seen with
the wild type
1S. Coexpression of
2a with
either one of the mutant
1S constructs had no effect on
the distribution of the
1S constructs (Table IV).
1S-Y366S and
1S-
351-380 remained
localized in the ER (Fig. 5, f and h). This
indicates that a specific interaction between
1S and
2a is involved in the
1S translocation
and that this interaction requires the intact
interaction domain in
1S.
Coexpression of
1a but Not of
2a
Enhances the Frequency of Ca2+ Current Expression--
The
rate at which tsA201 cells expressed Ca2+ current after
transfection with
1S alone or in combination with
1a or
2a was used to assess the
capability of
subunits to facilitate the insertion of functional
Ca2+ channels in the plasma membrane. Cells positively
identified as transfected by expression of GFP fluorescence were tested
for expression of Ca2+ currents with the whole-cell patch
clamp method using a voltage ramp protocol. High voltage-activated
Ca2+ currents were identified by an inward current peak at
potentials above +30 mV during the ramp depolarization (see
"Experimental Procedures" and Fig.
6a). Such Ca2+
currents were found in 14 of 40 (35%) tested cells co-transfected with
1S and
GFP and in 10 of 33 (30%) cells
co-transfected with
1S-Y366S and
GFP but in none of
25 cells co-transfected with
1S-
351-380 and
GFP.
When the
1S constructs were expressed without the
1a subunit, the expression frequency of high
voltage-activated currents was close to zero;
1S, 1 of
38 (2.6%) and
1S-Y366S, 0 of 46 (0%; Fig.
6a). The increased frequency of Ca2+ current
expression upon coexpression of
GFP suggests that the
1a subunit enhances current expression by
1S. Interestingly, this effect of
GFP was not limited
to the wild type
1S, the only
1S
construct that showed a structural association with the
1a subunit, but was also observed with
1S-Y366S. Thus, coexpression of the skeletal muscle
1a subunit significantly increased current expression by
the wild type
1S and the
1S-Y366S
(p < 0.001), whereas
1S-
351-380 did
not support expression of high voltage-activated current expression,
even in the presence of the
1a subunit. Due to the rare
occurrence of current expression in cells expressing
1S
without
1a, we did not attempt further analysis of
1a effects on current properties. The few cells analyzed
with a voltage-step protocol do not suggest differences in voltage
dependence, current kinetics, or current densities between cells
expressing
1S with and without
1a.

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Fig. 6.
Expression frequency of
high-voltage-activated Ca2+ currents in tsA201 cells
co-transfected with 1S or 1S-Y366S with
and without 1a ( GFP) or 2a (plus GFP).
a, expression of a high voltage-activated Ca2+
current was identified by an inward current peak above +30 mV during a
voltage ramp depolarization from 80 to + 70 mV (right
panel; see "Experimental Procedures"). Numbers give
the fraction of positive cells of the total number of cells tested.
Co-transfection with GFP caused a significant increase in current
expression compared with the poor expression without a subunit
(p < 0.001, left panel). The difference in
current expression between 1SY366S and
1SY366S plus 2a is not significant
(p > 0.05). b, representative current
traces during voltage steps from a holding potential of 80 mV to the
indicated test potentials (left panel) and the corresponding
peak current/voltage relationship (right panel). Data shown
in a, right panel, and b are from the
same cell co-transfected with a1S and GFP.
pA/pF, picoamperes/picofarads.
|
|
Since coexpression of the cardiac
2a subunit caused a
marked translocation of the skeletal muscle
1S subunit
to the plasma membrane (see above), we wanted to know how this affected
current expression in tsA201 cells. However, contrary to our
expectations, expression of Ca2+ currents was as poor and
not significantly different from that when
1S or
1S-Y366S was expressed without a
subunit (Fig. 6a). Currents were only found in 1 of 51 (2%) cells
transfected with
2a plus the wild type
1S
and in 4 of 43 (9%) cells transfected with
2a plus
1S-Y366S. Thus,
2a lacks the ability to
increase the frequency at which skeletal Ca2+ currents are
expressed in tsA201 cells. Apparently, the function of
2a responsible for the translocation of
1S to the plasma membrane (see above) is not coupled to
-induced stimulation of Ca2+ current expression.
 |
DISCUSSION |
In the present study we used heterologous expression of the
1S subunit of the skeletal muscle L-type
Ca2+ channel with and without the skeletal and cardiac
subunit isoforms to investigate the mechanisms and functions of their
interactions. A fusion protein of
1a and the green
fluorescent protein and
1S constructs with modifications
in the known
interaction domain in the cytoplasmic loop between
repeats I and II of
1S have been generated. The effects
of coexpressing these constructs on the subcellular distribution of the
channel subunits were analyzed with GFP- and
immunofluorescence-labeling, and the effects on expression of
Ca2+ currents were tested with patch clamp recordings. The
results indicate that two
subunit isoforms,
1a and
2a, that differ in their intrinsic ability to associate
with the plasma membrane also differ in their effects on the
subcellular distribution of the
1S·
complex and on
the expression of functional channels.
Subcellular Distribution of
1a and
2a--
The
1a subunit of the skeletal
muscle DHP-receptor or its GFP fusion protein (
GFP), expressed alone
in tsA201 cells, was localized diffusely throughout the cell. The
labeling pattern was similar to that of GFP alone, and no preferential
association with the plasma membrane or any other cellular structure
was seen. Thus, the hydrophilic
1a subunit appears to
lack a specific anchoring mechanism for the plasma membrane. This
localization was different from that of
2a, which was
preferentially localized in the plasma membrane when expressed alone in
tsA201 cells. An association with the plasma membrane has previously
been shown for
1b and
2a when expressed
without a
1 subunit in heterologous mammalian expression
systems (25, 27), and palmitoylation of two N-terminal cysteines was
suggested as possible anchoring mechanism of
2a (28).
Other
subunit isoforms, such as
3b and
4, were not localized in the plasma membrane but were
distributed throughout the cell when expressed in COS-7 cells (27).
Thus,
1a shares the diffuse distribution with
3b and
4. Even though the distribution pattern of
1a strongly suggests a cytoplasmic
localization, it does not rule out the possibility that
1a is, at least in part, associated with some diffusely
distributed membrane or cytoskeletal components. Significant amounts of
1a were found in the particulate fraction as well as in
the supernatant when cells were fractionated by centrifugation at
70,000 × g (not shown). This is consistent with
published data showing other
isoforms, including the non-plasma membrane-associated
3b and
4, in the
particulate fraction (27) and with data showing that the removal of the
membrane anchor of
2a did not change its distribution
between supernatant and particulate fraction (28). However, the
redistribution of
1a from a cytoplasmic localization to
the ER upon coexpression with
1S suggests that
1a is not tightly bound to other cytoplasmic structures
but is readily available for association with the
1S subunit.
Association and Functional Expression of
1a and
1S--
How do
1a and
2a,
which by themselves are localized in distinct cytoplasmic compartments,
interact with the
1S subunit, and how does this
interaction affect localization and expression of functional channels
in the membrane? The wild type and the mutant
1S
subunits of the skeletal muscle DHP receptor were all localized in a
tubular/reticular cytoplasmic membrane system, most likely the ER. No
plasma membrane association of
1S was observed with
immunocytochemistry, and essentially no skeletal-type Ca2+
currents were recorded in tsA201 cells transfected with any of the
1S constructs alone. The latter is concordant with the
generally observed poor current expression with the skeletal muscle
1S isoform in heterologous expression systems as
compared with other
1 subunit isoforms. Thus, without a
subunit, most of the expressed
1S was retained in
the ER, whereas only little or none was inserted into the plasma
membrane.
When
1a was coexpressed with the
1S
subunit, the subcellular distribution of
1a changed
dramatically, whereas the localization of
1S appeared
unaltered.
1a was now colocalized with the
1S subunit in the ER. The redistribution of
1a upon coexpression with
1S was blocked
when the
interaction domain in
1S had been mutated
(
1S-Y366S) or deleted (
1S-
351-380).
This indicates that the
1S subunit is sufficient for the
association of
1a with the DHP receptor complex and that
this interaction depends on the intact
interaction domain that has
been identified in the cytoplasmic loop between repeats I and II of all
examined
1 subunits (12, 13). In a parallel study in
dysgenic myotubes, we found that this stable
1S-
1a association is required for the
normal targeting of the
1a subunit but not of the
1S into the skeletal muscle triad (53). Thus, this
interaction is of importance in the native system. The fact that
1S and
1a associate with one another at
the ER shows that neither the processing of the
1S
subunit in the biosynthetic pathway nor the transport of
1S to the plasma membrane are necessary before the
formation of the
1S·
1a complex. Thus it
is possible that in the native system the association of
1S and
1a also occurs in the ER, which would be a prerequisite for a chaperon-like function of the
subunit
in the transport of
1S to the plasma membrane (27).
Indeed, upon coexpression of
1S and
1a,
Ca2+ currents could be recorded, indicating that
1a is required for the expression of functional channels
in the plasma membrane of transfected tsA201 cells. However, current
expression was not accompanied by detectable plasma membrane staining
of either
1S or
1a. Therefore, measurable Ca2+ currents can be supported by a concentration of
channels in the plasma membrane that is too low to be detected with
immunocytochemistry. The highest current densities recorded in our
experiments correspond to approximately 10 active
channels/µm2. Even if this number represents an
underestimate due to the possible presence of silent channels, the
expected density in the plasma membrane of the tsA201 cells would be
far below the density of approximately 2,200 channels/µm2
found in skeletal muscle triads where this channel can be reliably localized with the same antibody. Despite the fact that patch clamp
analysis gives evidence of channels in the plasma membrane that cannot
be visualized by immunofluorescence labeling, one can assume that these
channels show the same behavior with respect to association with
as
the channels observed in the ER, since both originate from the same
expression plasmids. This is supported by the finding that deletion of
the
interaction domain resulted in failure of both
1S·
1a complex formation and current
expression. Most interesting and unexpected, however, was the finding
that current expression was still induced when
1a was
coexpressed with the Y366S mutant
1S, which did not
stably associate with
1a. This differential behavior of
1S-Y366S with respect to stable association with
1a and current stimulation by
1a
indicates that the two properties of
1a are independent
of each other and that
1S·
1a complex
formation but not necessarily current stimulation utilizes the known
interaction domain in the I-II cytoplasmic loop of
1S.
Translocation of
1S upon Coexpression with
2a--
2a expressed alone was localized
in the plasma membrane of tsA201 cells. Its localization did not change
upon coexpression with
1S, but
2a
coexpression induced the translocation of the
1S subunit
from the ER to the plasma membrane. The
2a-induced translocation of
1S was only observed with the wild type
1S but not with mutants in which the
interaction
domain had been altered or deleted. This indicates that a specific
interaction of the membrane-bound
2a with the
interaction domain in the I-II cytoplasmic loop of
1S is
involved in incorporating the
1 subunit in the plasma
membrane. However,
1S and
2a were not precisely colocalized in the plasma membrane. Although
2a remained evenly distributed throughout the membrane,
1S was localized in aggregates of variable sizes and
shapes. Apparently,
2a facilitates the incorporation of
the
1S subunit into the plasma membrane but does not
remain tightly associated with
1S when this subunit forms aggregates. Even though
2a did not modulate
1S function, the ability of
2a to
reversibly bind to
1 subunits may be related to its
mechanism of modulation of other
1 subunit isoforms. For instance, it has been suggested that
competes with G-protein 
subunits for the binding site in the I-II cytoplasmic loop of neuronal
Ca2+ channel isoforms (45-47). For such a competition to
occur, free
and G-protein 
subunits must be available. A
subunit anchored in the plasma membrane like the G-protein 
subunits may have an advantage in such a competitive interaction over
subunits distributed in the cytosol. Our observation that
2a is uniformly distributed in the plasma membrane and
not necessarily concentrated in the
1S-containing
membrane patches is consistent with the existence of free
in the
plasma membrane of
1S-expressing cells.
A
-dependent translocation of the cardiac
1C
and the neuronal
1A subunits by membrane-bound
1b and
2a has been reported (25, 27, 28).
However, coexpression of these subunit isoforms also resulted in an
increase of current densities compared with cells expressing
1 subunits alone. This differs from the effects of
2a on the skeletal muscle
1S subunit that
did not lead to an induction of current expression. The structural
basis of such isoform-specific properties of a
subunit may be the
modulatory binding site in the N terminus of the
subunit (48, 49). Nevertheless, the cardiac
2a isoform promoted the plasma
membrane incorporation of
1S. Thus,
isoforms differ
in their ability to target the
1 subunit to the plasma
membrane as well as in their ability to functionally modulate the
1 subunit. Surprisingly, the targeting function and
induction of current expression are not necessarily linked.
1S current stimulation may be a specific property of the
1 isoforms, and plasma membrane localization of a
subunit may be important for quantitative incorporation of
1S into the plasma membrane. Then the combination of the
two properties, as seems to be the case for
1b, might be
the key for efficient expression of the skeletal muscle
Ca2+ channel in heterologous expression systems (30). This
idea is consistent with the findings of a recent study showing that expression of skeletal Ca2+ currents in Xenopus
oocytes is dramatically enhanced by coexpression of
1S
with
1b but not with
1a (50).
Differential
1-
Interactions--
Our present
results demonstrate that
1-
interactions affect both
membrane targeting and the induction of currents in an isoform-specific
manner. The structural changes induced by
1a and
2a, even though opposite in direction, were both
dependent on the intact
interaction domain in the cytoplasmic loop
between repeats I and II of the
1S subunit. In one case,
translocation of
1a to the ER failed; in the case of
2a, translocation of
1S to the plasma
membrane failed when the
interaction domain was mutated or deleted.
Thus the
interaction domain in the I-II cytoplasmic loop of
1S is critical for complex formation with different
subunit isoforms. Whether the
isoform is membrane-anchored or not
determines where in the cell the association occurs. In contrast, the
ability of the
1a subunit to induce expression of
Ca2+ currents is isoform-specific (
2a failed
to stimulate currents), and neither requires the intact
interaction
domain nor the stable association of
1S and
1a.
These differential effects of
1a and
2a
on the wild type and mutant
1S subunits can be explained
by two different models of
1S-
interactions. The
first model assumes that the
interaction domain in the I-II
cytoplasmic loop of
1S mediates all observed effects of
. This is supported by our observation that deletion of the
interaction domain blocks complex formation with either
isoform as
well as current stimulation upon coexpression of
1a. The
tyrosine-to-serin substitution of one of the nine conserved residues in
the
interaction domain may according to this model lower the
affinity enough to perturb complex formation without impeding the
interaction that is required for the expression of functional channels
in the membrane. However,
1S membrane targeting by
2a, which also required interactions with the intact
interaction domain, did not stimulate Ca2+ currents. This
shows that the association of a
subunit with
1S via
the conserved
interaction domain in the I-II cytoplasmic loop does
not lead to induction of Ca2+ currents. Therefore,
interactions with the
interaction domain in the I-II cytoplasmic
loop are necessary but not sufficient for
-induced current
stimulation. An alternative model, assuming the existence of a second
domain in
1S for isoform-specific functional interactions with
, could explain these observations. Whereas the
primary
interaction domain in the I-II cytoplasmic loop of
1S binds all known
subunits with high affinity (13,
51), a second
interaction domain may specifically interact with
certain
isoforms with a low affinity that by itself does not permit the stable association of the two subunits. In the native system, binding via the primary interaction domain may direct
1a
into position to functionally interact with the secondary binding site. In the heterologous expression system, high concentrations of
1a in the cytoplasm may allow this functional
interaction even without the tight association of the two subunits.
This model is supported by recent evidence for the existence of a
second binding site for the
subunits in the C terminus of the
1E subunit (52) as well as by evidence for
isoform-specific domains in the
subunit that are involved in
functional modulation of
1 subunits (48, 49).
In summary, the experiments reported here allow the distinction between
functional interactions of the Ca2+ channel
1S and
subunits and their complex formation.
Differences in the subcellular distribution of
subunits and the
occurrence of common as well as isoform-specific effects on the
skeletal muscle
1S subunit are indicative of the complex
regulation of Ca2+ channels by
subunits. Whether the
distinct types of
1S-
1a interactions are
mediated by one or more interaction domains of
1S
remains to be demonstrated.
We thank Drs. S. Froehner and J. Striessnig
for their generous gift of antibodies, Drs. T. Kamp, Y. Seino, and F. Döring for their generous gift of expression plasmids, and Dr. G. Eaholtz for the generous gift of tsA201 cells. We also thank Dr. H. Hoflacher for excellent technical assistance.