From the Institute of Molecular Pharmacology and
Biophysics and the ¶ Department of Cell Biology, Neurobiology and
Anatomy, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-0828
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ABSTRACT |
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In order to study the precise mechanisms of
1 subunit modulation by an auxiliary
subunit
of voltage-dependent calcium channels, a recombinant
3 subunit fusion protein was produced and introduced into oocytes that express the human
1C subunit.
Injection of the
3 subunit protein rapidly modulated the
current kinetics and voltage dependence of activation, whereas massive
augmentation of peak current amplitude occurred over a longer time
scale. Consistent with the latter, a severalfold increase in the amount
of the
1C subunit in the plasma membrane was detected by
quantitative confocal laser-scanning microscopy after
3
subunit injection. Pretreatment of oocytes with bafilomycin
A1, a vacuolar type H+-ATPase inhibitor,
abolished the increase of the
1C subunit in the plasma
membrane, attenuated current increase, but did not affect the
modulation of current kinetics and voltage dependence by the
3 subunit. These results provide clear evidence that the
subunit modifies the calcium channel complex in a binary fashion; one is an allosteric modulation of the
1 subunit
function and the other is a chaperoning of the
1 subunit
to the plasma membrane.
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INTRODUCTION |
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Voltage-gated calcium channels are heteromultimeric protein
complexes that consist of at least three (1,
2/
, and
) subunits and play a central role in
diverse biological functions such as excitation-contraction coupling,
excitation-secretion coupling, neurotransmitter release, and regulation
of gene expression. The
1 subunit accommodates the
channel pore, voltage sensor, and binding sites for various
channel-modifying compounds. It has also been shown that the basic
characteristics of the different channel types (L-, N-, P-, Q-, and
R-type) are carried by the corresponding
1 subunits (1).
Auxiliary subunits (
2/
,
, and
) modulate
calcium channel characteristics, such as current amplitude, voltage
dependence, and kinetics of activation and inactivation as well as
sensitivity to calcium channel antagonists (2-4).
The modulatory effects of the subunit on the Ca2+
channel complex have been extensively studied using coexpression of
cDNAs in heterologous systems. It is well established that
coexpression of the
1 subunit with a
subunit results
in an increase of peak current density (5), acceleration of activation
and inactivation kinetics, a leftward shift of the current-voltage
relationship, and increased dihydropyridine
(DHP)1 binding activity
(6-12). However, there have been inconsistencies in the reported
mechanism(s) by which these effects occur. Varadi et al. (6)
reported a 10-fold increase in the number of DHP-binding sites by
coexpression of the
1 subunit with the
subunit,
suggesting an increase in available channels within the plasma
membrane. In contrast, an increase in current amplitude without
affecting charge movement by
subunit coexpression was shown by
Neely et al. (8). These same authors later reported two
modes of activation of the
1C subunit (13). Coexpression
of a
subunit potentiated current by an increase of the
fast-activating component, an acceleration of the slow component, and a
larger proportion of long openings. An increase in DHP binding and
current density without a change in the amount of
1
subunit protein in the plasma membrane was reported by Nishimura
et al. (10). These reports suggest that the
subunit
modulates channel properties by "assisting" the
1
subunit in establishing a proper conformation suitable for a functional
Ca2+ channel, rather than affecting expression,
trafficking, or stability of the
1 subunit (reviewed by
Catterall (14)).
By using immunocytochemical methods, Chien et al. (15)
showed that the 2a subunit acts as a chaperone-like
molecule to facilitate membrane targeting of the
1C
subunit, without affecting the total amount of expressed
1C subunit in human embryonic kidney cells. Coexpression
of the
1C subunit with
2a resulted in a marked increase in localization of the
1C subunit to the
plasma membrane. These observations were confirmed by Brice et
al. (16) who coexpressed the
1A with
2/
and several different
subunits. By using
Xenopus oocytes as an expression system, Shistik et
al. (17) reported opposite results in that no change in the amount of
1 subunit protein was found in the plasma membrane
when coexpressed with a
subunit. Interestingly, an increase in
charge movement has been reported when
1C and
subunits were coexpressed in a mammalian cell system (18, 19). These
discrepancies have been attributed to differences in expression systems
employed (20). Taken together, the mechanisms by which the
subunit modulates calcium channel activity remain unclear. The limitation of
previous studies are that they were all done using coexpression of the
1 and
subunits, which makes it difficult to answer
the question as to which level of the biosynthesis of the channel complex is the
subunit working, i.e. translation,
trafficking, and/or direct binding to the membrane-incorporated
1 subunit.
We address these questions by injecting a recombinant subunit
protein into Xenopus oocytes expressing the
1C subunit and observing the time course of modulation.
We found that changes in voltage dependence of activation and kinetics
occurred at an earlier stage in the time course, compared with
increases in current amplitude which required a substantially longer
time to reach plateau. This suggests that the effects are functionally
uncoupled and occur by distinct mechanisms. By using confocal
laser-scanning microscopy (CLSM), we found that the amount of the
1 subunit in the plasma membrane was substantially
increased by the
subunit. Furthermore, pretreatment of oocytes with
bafilomycin A1, a vacuolar type H+-ATPase
(V-ATPase) inhibitor (21), inhibited the
subunit effect on current
amplitude, abolished the increase in channel amount in the plasma
membrane, but did not influence the
effects on voltage dependence
and current kinetics.
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EXPERIMENTAL PROCEDURES |
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Bacterial Production and Purification of 3 Subunit
Fusion Protein--
The human calcium channel
3
cDNA clone (22) was subcloned into pBluescript SK(+) between the
HindIII and BamHI sites. This plasmid was cleaved
with HindIII, and the protruding end was filled in with T4
DNA polymerase and then cut with BamHI to liberate the
3-coding fragment. The pET15(b) vector (Novagen) was
cleaved with NdeI, filled in with T4 DNA polymerase, and
then cut with BamHI. Finally, the
3 fragment
was ligated into the blunt end/BamHI sites. This strategy
has generated an in-frame cloning of the
3 protein with
the His6-tag sequence and resulted in a fusion product that
had the following peptide fused to the N-terminal sequence of the human
3 protein, MGSSHHHHHHSSGLVPRGSHKLDP. The sequence of the
construct was verified by sequencing through the junction regions.
In Vitro Translation of the h3 Subunit and Binding
of GST Fusion Proteins--
The I-II, II-III, and III-IV
intracellular loops were extracted from the human heart calcium channel
cDNA (23) via PCR. By using PCR we performed site-directed
mutagenesis, placing unique BamHI and XhoI
restriction sites at both the 5' and 3' ends of these fragments. The
PCR products containing the I-II loop (nucleotides 1089-1499), the
II-III loop (nucleotides 2133-2633), and the III-IV loop
(nucleotides 3402-3574) were subcloned into the pGEX-4T-1 vector
(Amersham Pharmacia Biotech), which is under the control of the
tac promoter and contains the glutathione
S-transferase (GST) tag. The resulting subclones, pGEXI-II,
pGEXII-II, and pGEXIII-IV, were sequenced, and analysis confirmed the
presence of the intracellular loops and the absence of additional
mutations. The pGEX-4T-1, pGEXI-II, pGEXII-III, and pGEXIII-IV
plasmids were transformed into Novablue (DE3) cells (Novagen).
Production and purification of GST fusion proteins were done according
to the manufacturer's protocol.
Electrophysiology--
Xenopus laevis oocytes (stage
V-VI) were prepared as described previously (26). Capped cRNA was
synthesized from XbaI-linearized human heart L-type calcium
channel 1 subunit DNA template (23) and injected into
the oocytes (50 nl, 0.2 µg/µl). After 4-5 days of incubation at
19 °C in the solution containing (in mM) 96 NaCl, 2 KCl,
1 MgCl2, 1.8 CaCl2, 5 HEPES, 2.5 sodium
pyruvate, 0.5 theophylline, pH 7.5, supplemented with 100 units/ml
penicillin and 100 µg/ml streptomycin, oocytes were injected with 50 nl of either
3 subunit fusion protein solution (90 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 10 mM EDTA, 340 ng/µl protein, pH 7.4, with Tris)
or vehicle (same composition except protein). The final concentrations
in the oocytes were 300 nM
3 subunit fusion
protein, 4.5 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 0.5 mM EDTA, assuming the volume of oocytes was 1 µl. We found this was a saturating amount of protein since in our
preliminary experiments higher concentrations did not result in greater
effects. In the experiments indicated, oocytes were preincubated in a
solution containing 500 nM bafilomycin A1 for
2-3 h prior to the injection of the protein. After injection, they
were further incubated in the presence of bafilomycin A1
until currents were measured. Bafilomycin A1 was dissolved
in dimethyl sulfoxide as a 1 mM stock solution. The final
concentration of dimethyl sulfoxide was 0.05%, which had no effect on
the currents or the
effect.
Immunofluorescence Studies--
DNA sequence complementary to
the YPYDVPDYA epitope sequence of influenza virus hemagglutinin (HA)
(27), which is recognized by the 12CA5 mouse monoclonal antibody, was
introduced to the N terminus immediately after the initiator methionine
of human heart L-type calcium channel 1C subunit DNA by
PCR-based mutagenesis. The cRNA of the epitope-tagged
1
subunit was transcribed and injected into X. laevis oocytes
to express tagged channels. The functional characteristics of HA
epitope-tagged
1 were tested in a separate set of
experiments, and we found that the expressed currents and modulation of
them by the
3 subunit were indistinguishable from those
of wild type (data not shown). After 4-5 days of incubation, oocytes
were injected with the
3 subunit fusion protein as
described above. Four hours after injection, oocytes were fixed with
3.7% formaldehyde, 0.25% glutaraldehyde, and permeabilized with 0.2% Triton X-100 at room temperature. Oocytes were then post-fixed in 100%
methanol at
20 °C overnight, incubated with 2% bovine serum
albumin for 1 h at room temperature, followed by incubation with
10 µg/ml fluorescein-conjugated anti-HA monoclonal antibody (Boehringer Mannheim) at 4 °C overnight. Oocytes were then washed extensively in PBS and examined by CLSM.
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RESULTS |
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Production and Purification of a Recombinant Human 3
Subunit Fusion Protein--
The human
3 subunit fusion
protein was purified from the soluble fraction of a bacterial culture
induced with IPTG at 23 °C for 8 h. The soluble fraction was
then applied to a Ni2+-agarose column. Approximately 88.5%
of total protein was in the flow-through. A smaller amount of total
protein (~8%) weakly bound to the resin and was washed out by 60 mM imidazole. Approximately 3.5% of the total protein
bound firmly to the Ni2+-agarose and was eluted with 1 M imidazole and 0.5 M NaCl (Fig. 1A).
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In Vitro Binding of the 3 Subunit to the I-II
Intracellular Loop--
To determine whether the recombinant
His6-tagged
3 subunit was able to interact
with the calcium channel intracellular I-II loop, as identified by
Pragnell et al. (28), we created GST fusion proteins of the
I-II, II-III, and III-IV loops of the
1C subunit and
screened with an in vitro transcribed and translated, 35S-labeled His6-tagged
3
subunit. The data from these experiments clearly show that the
His6-tagged
3 subunit is able to interact with the I-II loop in a highly specific manner (Fig.
2A). In addition, these
results indicate that the
3 subunit did not interact
with the control GST, II-III, and the III-IV loops. A sample of the eluate was run on an 4-15% SDS-PAGE gel and stained with Coomassie Blue to determine whether the purified fusion protein could also be
eluted from the glutathione-Sepharose. All four GST fusion proteins
were purified and present in the binding assay eliminating the
possibility of nonspecific interaction with other protein components
(Fig. 2B). These experiments confirm that the
His6-tagged
3 subunit maintains its ability
to interact with the I-II loop of the
1C subunit
in vitro.
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Electrophysiological Properties of Expressed Human
1C Subunit in Xenopus Oocytes and
Time-dependent Effects of Injected Human
3
Subunit Fusion Protein--
Oocytes were injected with human
1C subunit cRNA and incubated for 4-5 days. The control
(
1C subunit alone) Ca2+ channels expressed
in oocytes showed peak barium currents of 149 ± 14 nA
(n = 14) when depolarized from a holding
potential of
80 mV to test potentials between
30 and +60 mV. The
control current exhibited a slow activation and very little
inactivation (Fig. 3A), in
agreement with previous studies (2, 9). The threshold potential for
current activation was found between
20 and
10 mV, and the current
peaked at +40 mV. After injection of the
3 subunit
fusion protein, an increase in current amplitude, a change in the
voltage dependence of activation, and changes in activation and
inactivation kinetics occurred in a time-dependent manner
(Fig. 3A). After injection of the
3 subunit
fusion protein the activation threshold shifted to hyperpolarizing
potentials between
30 and
20 mV, and the current peaked between +20
and +30 mV. The current-voltage (I-V) relationships corresponding to
the current traces depicted in Fig. 3A are superimposed in Fig. 3B. Most notably, the shift of the I-V curve occurs
within 2 h, whereas the increase in current amplitude requires a
longer time scale.
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Effects of 3 Subunit Fusion Protein on Peak Current
Amplitude and the Influence of Bafilomycin A1
Treatment--
Fig. 4 depicts the
average time course of peak Ba2+ current amplitude
enhancement after injection of the
3 subunit fusion
protein. We observed an increase in peak current amplitude, 2.3-fold at 1 h and 2.9-fold at 2 h after injection of the
3 subunit. However, more than half of the increase
occurred after 2 h, reaching a plateau at 3 h (6.5- and
6.6-fold for 3 and 4 h after injection, respectively). The effect
of the
subunit on the current amplitude is clearly slower than the
effect on the voltage dependence of activation (cf. Fig.
5). The time of the half-maximal effect
for the increase in current amplitude was between 2 and 3 h.
Injection of the vehicle had no significant effect on peak current
amplitude, although after 4 h the current amplitude appeared to be
smaller in some cases. We attribute this to a nonspecific mechanistic disruption by the injection itself.
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Effects of 3 Subunit Protein on the Voltage
Dependence of Activation--
Steady-state activation curves were
derived from I-V relationships, and the
3 subunit
effect on the voltage dependence of activation was further analyzed
(Fig. 5A). Control currents showed a shallow activation
curve with a half-activation potential of 29.9 ± 1.5 mV
(n = 14). Injection of the
3 subunit
protein caused a leftward shift of the curve and an increase in the
slope. Most dramatic changes occurred within 1 h, and no
significant changes were observed between 2, 3, and 4 h after
injection. As shown in Fig. 5B, ~70% of the negative
shift in the half-activation potential occurred within 1 h after
injection, reaching a plateau at 2 h. The time of the half-maximal
effect was less than 1 h. The vehicle had no effect on the voltage
dependence of activation. Bafilomycin A1 treatment did not
influence the ability of the
3 subunit to shift the
half-activation potential in the negative direction (Fig.
5B).
Effects of the 3 Subunit Protein on Current
Kinetics--
We measured the time to half-peak current as a parameter
of the macroscopic activation kinetics. As shown in Fig. 3A,
the control (
1 subunit alone) current showed slow
activation. When depolarized to +30 mV from a holding potential of
80
mV, the time to half-peak was 19.2 ± 1.0 ms (n = 9) (Fig. 6A). The
injection of oocytes with the
subunit protein rapidly facilitated
activation kinetics, reaching a plateau within 2 h, with the time
of the half-maximal effect occurring within 1 h. The vehicle did
not change activation kinetics. Pretreatment of oocytes with
bafilomycin A1 did not change activation kinetics of
control (
1 subunit alone) currents and did not modify
acceleration of activation kinetics by the
subunit protein (Fig.
6A).
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Subunit Protein Increases the Amount of
1
Subunit in the Plasma Membrane--
We tested whether the injection of
the
3 subunit protein into oocytes promoted
1C protein delivery to the plasma membrane. The HA
epitope-tagged
1C subunit was expressed in
Xenopus oocytes that were then injected with the
3 subunit protein. We found no difference in either the
characteristics of Ba2+ current or effects of the
3 subunit protein between the wild-type channels and
epitope-tagged channels. The epitope-tagged
1C subunits were detected in the plasma membrane immunocytochemically using CLSM
and quantitated by measuring the pixel intensity. When the epitope-tagged
1C was expressed alone in
Xenopus oocytes, we observed very little fluorescence in the
plasma membrane. In the presence of the
3 subunit
protein, the amount of the
1C subunit in the plasma
membrane increased severalfold (Fig. 7).
Furthermore, pretreatment of oocytes with bafilomycin A1
completely abolished this increase, strongly suggesting that this
process is dependent on protein translocation. We obtained similar
results in 10-15 oocytes for each group over two different batches of
oocytes.
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DISCUSSION |
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Modulation of calcium channel activity by auxiliary subunits has
been extensively studied using many different recombinant expression
systems. These studies have clearly demonstrated that coexpression of
the subunit alters calcium channel characteristics by increasing
current density, shifting the voltage dependence of activation and
accelerating channel kinetics. Taken together, these modulatory
alterations are believed to impart the inherent current characteristics
observed in native preparations. An issue that remains unresolved is
whether the
subunit increases the amount of the
1
subunit in the plasma membrane or modulates calcium channel functions
solely through an allosteric pathway. Experimental observations to date
present conflicting results. When the
1 subunit was
coexpressed with the
subunit in Xenopus oocytes, no
change in channel expression was observed, when measured as a function
of gating charge movement (8). Moreover, in the same experimental
system, the amount of 35S-labeled
1 subunit
in the plasma membrane did not change (17). Furthermore, Nishimura
et al. (10) have also shown no change in the
1 subunit content of membrane fractions by
immunoblotting analysis of cells transfected with the
subunit. In
contrast, several studies using mammalian cells have shown an increase
in charge movement when transfected with
(18, 19) and the
involvement of the
subunit in translocation of the
1
subunit to the membrane (15, 16). However, the biochemical mechanism(s)
responsible for this modulation have remained unclear due to
insufficient experimental methods to monitor time-dependent
biological events occurring intracellularly. In an effort to overcome
this limitation, and to resolve apparent conflicting data, we expressed
the L-type calcium channel
1 subunit in oocytes, and
after injecting a highly purified recombinant
3 subunit
protein, we examined the time-dependent changes to peak
current, voltage dependence, and kinetics. Our results demonstrate for
the first time a time-dependent uncoupling of
3 subunit modulation of voltage dependence and kinetics
from enhancement of peak current density. Moreover, these results
suggest that
subunit modulation occurs via an allosteric mechanism
and through facilitation of protein translocation.
3 Subunit Binding to the I-II Intracellular
Loop--
In order to determine whether the His6-tagged
3 subunit interacts with the
1
interaction domain (AID) of the intracellular I-II loop as described
by Pragnell et al. (28), we screened GST fusion proteins
containing each of the three intracellular loops I-II, II-III, and
III-IV with an 35S-labeled His6-tagged
3 subunit. We found that the
3 subunit was able to interact with the I-II loop, suggesting that the presence of six histidine residues at N terminus does not interfere with the
binding of this subunit to its intracellular binding site. Since Walker
and co-workers (31) have shown that the modulatory functions of the
subunit are largely dependent on this interaction, and we have shown
that the His6-tagged
3 subunit can interact with the I-II loop, the
3 subunit should modulate
calcium channel function when injected into oocytes as a purified
fusion protein. It was also clear from the results of these experiments
that the
3 subunit did not interact with either the
intracellular II-III or the III-IV loops. Although our in
vitro experiment showed highly specific interaction between the
3 subunit and the
1C I-II loop, we
cannot exclude possible weak or transient interactions with other
intracellular regions.
Injection of Oocytes Expressing the Ca2+ Channel
1 Subunit with a Recombinant
Subunit Fusion Protein
Induces Effects Comparable to
1-
Coexpression--
Effects of
subunit coexpression on the
1C subunit have been studied using Xenopus
oocytes (7, 8, 17, 32, 33) as well as in mammalian cells (6, 9, 10, 15,
18). It is now generally accepted that coexpression of the
subunit results in a 2-fold to more than 100-fold increase in peak current amplitude, a hyperpolarizing shift of the voltage dependence of activation, and acceleration of kinetics of the current (to a different
extent, depending on the different types of
subunits). Since our
results showed comparable effects of the
subunit on these
parameters (e.g. ~6.5-fold increase in current amplitude, ~16 mV hyperpolarizing shift of half-activation potential, and acceleration of activation and inactivation kinetics) within 3 h
of
subunit protein injection, it is unlikely that the
subunit is enhancing protein synthesis of
1 subunits in the
endoplasmic reticulum. Employing our experimental conditions, it takes
at least 3 days from the time of co-injection of the
1C
and
subunit cRNAs and at least 4 days for the
1C
subunit cRNA alone to get measurable current through these expressed
channels. Therefore, even if we assume that the presence of the
subunit somehow facilitates protein synthesis of the
1
subunit, its contribution to the observed effects should be minimal,
since our recording time scale is shorter. Thus, we believe that the
effects of the
subunit are exerted mainly on mechanism(s)
downstream of protein synthesis.
The Time Course of the Subunit Effects Can Be Categorized in
Two Distinct Patterns--
In our present study, we analyzed the time
course of four parameters after injection of the
subunit protein as
follows: 1) peak Ba2+ current amplitude; 2) voltage
dependence of activation; 3) activation kinetics; and 4) inactivation
kinetics. Among these, only the peak current amplitude increased within
a slow time framework; it took 3 h to reach plateau and the time
of the half-maximal effect was between 2 and 3 h. The other three
parameters behaved very similarly to each other and changed with a
faster time course, reaching a steady level in 2 h, and the time
of the half-maximal effect was less than 1 h. According to the
in vitro binding study by De Waard et al. (24),
AID and the
1b subunit bound at a rate constant of 0.1 min
1·µM
1, and when using
500 nM AID, the rate constant corresponded to a half-time
of about 20 min. Since our "faster" time course falls within the
same time range as their results, we believe that the faster time
course indicates direct binding of the injected
3 subunit protein to the
1C subunits that already exist in
the plasma membrane. We do not know the exact time it takes for the injected
3 subunit protein to diffuse, reach the plasma
membrane, and build to a saturated concentration. However, since an
excess amount of
3 subunit protein was injected (final
concentration in the oocyte cytoplasm was 300 nM, which is
about 5.4 times higher than the reported Kd of AID
and
3 subunit by De Waard et al. (24), 55.1 nM), we assume it takes less than 1 h. Taken together,
it seems that the binding of the injected
3 subunit protein to the
1C subunits in the plasma membrane
reaches equilibrium within 2 h. Bafilomycin A1
treatment did not influence the
effects that are categorized with a
faster time course but did abolish the increase of
1
subunit by the
subunit. This finding also supports the concept that
allosteric modulation of
1 subunits in the plasma
membrane by the
subunit is responsible for the faster effects,
whereas the "slower" component implies the existence of a distinct
mechanism for modulation. In order to clarify this, we addressed the
question whether the
subunit modulation of current amplitude occurs
during protein translocation.
Influence of the Pretreatment with Bafilomycin
A1--
Bafilomycin A1, a V-ATPase inhibitor,
inhibits intracellular glycoprotein transport by impairing the
acidification of organelles (30). In the presence of this compound the
subunit failed to increase the amount of
1 subunit
in the plasma membrane. Bafilomycin A1 also significantly
blocked the effect of the
subunit on current amplitude. The results
strongly suggest that the effect of the
subunit protein on current
amplitude is largely dependent on intracellular translocation of
nascent
1 subunits. The
subunit elicited a
~1.8-fold increase of current when oocytes were treated with
bafilomycin A1, despite a complete loss of increase of the
1 subunit in the plasma membrane. An attractive
explanation for the current increase is that the injected
subunit
allosterically modulates the population of
1 subunits
that are already inserted in the membrane. This is in agreement with
single channel analyses done by Wakamori et al. (33), in
which the coexpression of the
1 subunit with the
subunit resulted in ~2-fold increase in the channel open
probability.
Possible Role of Xenopus Oocyte Endogenous Subunit--
Recently Tareilus et al. (25) cloned an
endogenous
subunit from Xenopus oocytes
(
XO) which is highly homologous to the mammalian
3 subunit. Injection of Xenopus
antisense
oligonucleotides significantly reduced the current through the
expressed
1C and
1E subunit of the
Ca2+ channel. Based on this, the authors proposed that the
"
1 alone" channels are in fact forming an
1 subunit-endogenous
subunit complex
(
1
XO). However, it is not possible at
this time to determine whether the endogenous
XO was
present in high enough concentration to complex with the
1 subunit in the absence of exogenous
. Thus, it
seems likely that our observations incorporate the effects of exogenous
3 subunit on the
1
XO
complex. However, the basic characteristics of the control
(
1 alone) current in the present study, viz.
slow activation, slow inactivation, smaller current amplitude, and a
shifted voltage dependence to the depolarized direction, and the
modulation of these parameters by the application of exogenous
subunit are in good agreement with previous studies using a variety of
mammalian cells (6, 9, 10, 15, 19) in which no endogenous
subunits
have been reported.
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ACKNOWLEDGEMENTS |
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We thank Gabor Mikala for making the HA
epitope-tagged 1 subunit, Ilona Bodi for oocyte
preparation, and Udo Klöckner and Howard Motoike for helpful
discussions.
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FOOTNOTES |
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* This work was supported in part by the Naito Foundation (to H. Y.), by a fund from Kansai Medical University (to M. H.), and by National Institutes of Health Grant P01 HL22619-19 (to A. S.).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.
§ Supported by Postdoctoral Fellowship SW-97-35-F from the American Heart Association Ohio-West Virginia Affiliate.
To whom correspondence should be addressed: Institute of
Molecular Pharmacology and Biophysics, University of Cincinnati College of Medicine, 231 Bethesda Ave., P. O.Box 670828, Cincinnati, OH 45267-0828. Tel.: 513-558-2466; Fax: 513-558-1778; E-mail:
varadig{at}email.uc.edu.
1
The abbreviations used are: DHP,
dihydropyridine; CLSM, confocal laser-scanning microscopy; V-ATPase,
vacuolar type H+-ATPase; IPTG, isopropyl
-D-thiogalactopyranoside; GST, glutathione S-transferase; AID,
1 interaction domain;
PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered
saline; PCR, polymerase chain reaction.
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REFERENCES |
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