(Received for publication, June 23, 1995; and in revised form, August 24, 1995)
From the
Cardiac L-type Ca channels are multisubunit
complexes composed of
,
, and
subunits. We tested the roles of these subunits in
forming a functional complex by characterizing the effects of subunit
composition on dihydropyridine binding, its allosteric regulation, and
the ability of dihydropyridines to inhibit channel activity.
Transfection of COS.M6 cells with cardiac
(
) led to the appearance of dihydropyridine
([
H]PN200-110) binding which was increased by
coexpression of cardiac
(
),
(
), and the
skeletal muscle
. Maximum binding was achieved when cells
expressed
,
, and
. Cells
transfected with
and
had a binding affinity
that was 5-10-fold lower than that observed in cardiac membranes.
Coexpression of
normalized this
affinity.(-)-D600 and diltiazem both partially inhibited
PN200-110 binding to cardiac microsomes, but stimulated binding in
cells transfected with
and
. Again, coexpression
of
normalized this allosteric regulation. Therefore
coexpression of
and
completely
reconstituted high affinity dihydropyridine binding and its allosteric
regulation as observed in cardiac membranes. Skeletal muscle
was
not required for this reconstitution. Expression in Xenopus oocytes demonstrated that coexpression of
with
increased the potency and maximum extent of
block of Ca
channel currents by nisoldipine, a
dihydropyridine Ca
channel antagonist. Our results
demonstrate that
subunits are essential components of
the cardiac L-type Ca
channel and predict a minimum
subunit composition of
for this
channel.
Voltage-gated L-type Ca channels are a key
element in the excitation-contraction coupling in cardiac muscle. These
channels are the molecular target for many drugs including the
clinically useful nifedipine, verapamil, and diltiazem. Physiologically
they represent one of the sites where
-adrenergic stimulation
regulates cardiac function. Thus, understanding the molecular structure
of cardiac L-type Ca
channels, including their
subunit composition and the structure-function relationship of each
subunit, is of fundamental importance. Biochemical purification of
L-type Ca
channels from skeletal muscle has revealed
that, in this tissue, the Ca
channel complex consists
of five subunits(1) , which are encoded in four genes (2, 3, 4, 5, 6) .
Functional expression of
in cells lacking endogenous
Ca
channel subunits has established that the
subunit is by itself able to form a functional
channel(7) , although the activation kinetics of the channel
formed by
alone is abnormal. Coexpression of the
skeletal muscle
subunit normalizes the activation
kinetics(8) , suggesting that
is an essential component
of the skeletal muscle L-type channel.
Definitive identification of
the subunit composition of the cardiac L-type Ca channel has been hampered by the low abundance of dihydropyridine
binding sites in cardiac muscle. Biochemical studies have demonstrated
the existence of
,
, and
-
in heart(9, 10, 11, 12) . This has
been further confirmed by molecular cloning
approaches(3, 13, 14, 15) . Cardiac
and its splice variants, collectively referred to as
(16) , have been studied extensively by
expression in Xenopus oocytes and mammalian cells.
Coexpression studies have suggested crucial physiological roles for the
subunit in the functioning of the cardiac Ca
channel as in the case of the skeletal muscle channel. In these
studies, various
subunits are found to increase peak current,
accelerate activation kinetics, and shift the voltage dependence of
activation to more hyperpolarized
potentials(15, 17, 18, 19) . The
mechanism by which
increases current density is ascribed to a
facilitated coupling between the movement of voltage sensor and pore
opening without affecting the number of channels being
expressed(20) .
subunits have also been found to
facilitate dihydropyridine binding to the channel by increasing the B
of receptor sites or the affinity for
ligands(21, 22) . In contrast, the effect of
on cardiac
has not been consistent,
although stimulation of peak current (13, 23) and
changes in inactivation kinetics (17, 21) have been
reported. One problem in dealing with these variable effects is that
there have been no good criteria as to whether these effects are
required for the physiological function of the channel or they are
artifactual effects observed only in heterologous expression systems. A
similar confusion surrounds the role of the skeletal muscle
,
which is able to affect the cardiac isoforms of
(17, 18) although its expression has been
detected only in skeletal muscle(5, 6) .
In this
article, we describe an analysis of the functional importance of
cardiac Ca channel subunits by characterizing their
effects on the ligand binding properties of the complex. By transiently
expressing a cardiac
in various combinations with a
cardiac
,
, and the skeletal muscle
in
COS.M6 cells, we demonstrate that high affinity dihydropyridine binding
and its allosteric regulation are identical to those in cardiac
membranes only when
,
, and
are expressed, and that
plays a key role in the formation of cardiac L-type Ca
channel complex.
(+)-[H]PN200-110
(isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridinecarboxylate),
specific activity 80-85 Ci/mmol, was purchased from Amersham.
Nisoldipine was a kind gift from Dr. David J. Triggle (State University
of New York at Buffalo). Nitrendipine,(-)-D600, and diltiazem
were purchased from CalBiochem. Tissue culture media and sera were
purchased from BRL/Life Technologies, Inc. NuSerum was purchased from
Collaborative Research (Bedford, MA). T7 RNA polymerase and other
chemicals for in vitro transcription were obtained from
Boehringer Mannheim.
The transfection efficiency of this procedure was tested
by transfecting cells with 2 µg of the pSV-Gal indicator
plasmid and testing cells histochemically for the functional expression
of
-galactosidase as described by MacGregor et
al.(26) . Briefly, 60 h after transfection, the cells in a
100-mm dish were rinsed with 10 ml of phosphate-buffered saline, and
overlaid with 1 ml of 0.6% glutaraldehyde in phosphate-buffered saline
for 10 min at room temperature. The cells were then rinsed twice with
10 ml of 50 mM Tris (pH 7.5) and 150 mM NaCl (TBS)
and overlaid with 8 ml of TBS containing 0.5 mg/ml
5-bromo-4-chloro-3-indoyl
-D-galactoside, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and
2 mM MgCl
. After an overnight incubation at 37
°C, the proportion of cells developing blue color due to
-galactosidase expression was determined with a phase-contrast
microscope at a magnification of 200
. The transfection
efficiency thus tested was between 30 and 40%.
Currents were
recorded using the cut-open oocyte vaseline-gap voltage clamp technique (28) with a CA-1 amplifier from Dagan Corp. An oocyte was
placed in triple Perspex chambers that isolated a portion
(approximately one-sixth to one-fifth) of the oocyte surface for
current recording. Oocyte membrane exposed to the bottom compartment
was permeabilized by a brief treatment with 0.1% saponin to allow low
access resistance to the interior of the oocyte. The voltage pipettes,
filled with a solution containing 2.7 M sodium
methanesulfonate, 10 mM EGTA, and 10 mM NaCl, had tip
resistance of 300 to 400 k. Data acquisition and analysis were
performed using the pCLAMP system (Axon Instruments). Currents were
induced by 250-ms depolarization steps from a holding potential of
-40 mV. Linear components were subtracted using P/-4
protocol. Current signals were filtered at 0.5 kHz and digitized at 2
kHz.
All experiments were performed at room temperature (22-24
°C). Each oocyte was injected with 50 nl of 50 mM
BAPTA(Na)(
)prior to recording to minimize the
contamination by the oocyte endogenous Cl
currents,
which can be activated by the influx of Ba
as well as
of Ca
(29) . The external recording solution
contained 10 mM Ba
, 96 mM Na
, and 10 mM HEPES, pH adjusted to 7.4
with methanesulfonic acid. The internal solution contained 120
mM potassium glutamate, 10 mM EGTA, and 10 mM HEPES, pH adjusted to 7.3 with KOH. Nisoldipine was diluted in the
external solution so that 5% of the top chamber volume was replaced to
achieve the desired final concentration. Before an oocyte was subjected
to nisoldipine, it was observed for 10-15 min with repeated
stimulation to ensure a stable current amplitude. Under our conditions,
currents usually remained stable for at least 1 h. Run-down occurred
only in oocytes which were severely damaged and these oocytes were
discarded. In order to obtain cumulative dose-response relationships
for nisoldipine, an oocyte was stimulated by a 250-ms depolarization
step to +10 mV repeated every 10 s. A higher drug concentration
was applied after the response to the previous concentration
stabilized, usually within 20 s.
Fig. 1shows
(+)-[H]PN200-110 binding to membranes from
COS cells transfected with various combinations of subunits. The
results represent the average from four experiments. Cells transfected
with
only had a measurable binding of 3.8 ±
1.5 fmol/mg of protein. Thus, the level of binding achieved in cells
transfected with
alone was very low and close to the
limit of detection. Coexpression of
with
,
, or
increased the amount of
(+)-[
H]PN200-110 binding. The most effective
subunit was
, which stimulated binding 19-fold. In comparison,
stimulated binding only 4.4-fold.
appeared to
have a small effect (an 1.5-fold increase) although this effect may not
be significant due to the sensitivity of the binding assay.
Co-transfection of
with combinations of
,
, and
revealed interesting properties in terms
of the effects of these subunits in stimulating binding. First, maximum
PN200-110 binding was obtained when
,
, and
were coexpressed; binding reached 165.4 ± 8.0
fmol/mg of protein which represents a 44-fold stimulation. This degree
of stimulation by
and
together is much greater
than predicted by the sum of the effects of each subunit alone,
suggesting that
and
functioned synergistically.
Second, coexpression of
with
and
stimulated binding by 19-fold, coexpression with
stimulated binding by 5.8-fold, and coexpression with
stimulated binding by 39-fold. These
results suggest that
did not have a significant effect on
dihydropyridine binding when either
or
or both
were coexpressed with
.
Figure 1:
Comparison of specific
(+)-[H]PN200-110 binding to membranes from
COS cells transfected with Ca
subunits in various
combinations. Binding assays were performed using 0.4 nM (+)-[
H]PN200-110, 0.4 mM Mg
, and 1 mM Ca
. Bars indicate standard error of mean (n =
3-4).
The stimulation of
dihydropyridine binding to by other subunits may be
due to an increase in the affinity for ligand or due to an increase in
the number of receptors. In order to investigate these possibilities,
(+)-[
H]PN200-110 binding was measured at
various concentrations of the ligand and Scatchard analyses were
performed. COS cells expressing
alone,
with
and/or
but without
had amounts
of PN200-110 binding that were too low to allow for Scatchard analysis.
Therefore binding in cells expressing
was used
to determine the effects of other subunits. Binding to cardiac
microsomal membranes was carried out in parallel as control. Binding
was also compared in the absence and presence of 1 mM added
Ca
in all membrane preparations to determine the
dependence on divalent cations. All data represent the average from
three to four experiments each performed in duplicate.
[
H]PN200-110 binding to rabbit cardiac microsomal
membranes had a K
of 38 ± 4 pM (n = 3) and 33 ± 6 pM (n = 3) in the presence and absence of Ca
,
respectively. The B
values were 213 ± 14
and 176 ± 11 fmol/mg of protein in the presence and absence of
Ca
, respectively (Table 1). The K
values obtained in the present study were
similar to those reported previously for cardiac myocytes(27) .
The results demonstrated that high affinity dihydropyridine binding to
cardiac membranes did not require added Ca
. In
contrast, PN200-110 binding to membranes from COS cells expressing
was different in two ways. First, in the
presence of Ca
, the K
of
expressing cells was 175 ± 11
pM; this reflects a 5-fold lower affinity than that in cardiac
membranes. Second, the affinity was still lower in the absence of added
Ca
. Both of these differences suggest that additional
subunits may be required to reconstitute the high affinity
dihydropyridine binding seen in cardiac membranes.
Fig. 2shows representative Scatchard plots of binding in the
presence of Ca in COS cells expressing either
,
,
, or
. In the presence of
Ca
, coexpression of
,
, or both
with
increased the affinity of PN200-110 binding
in comparison with cells expressing
only. The K
values (Table 1) were very similar to that
in cardiac microsomal membranes. However, in the absence of added
Ca
only in cells expressing
and
, the affinity of PN200-110
resembled that in cardiac membranes. Binding affinity in cells
expressing
was 6.5-fold lower than that in
cardiac membranes. Thus, coexpression of
with
failed to correct the divalent cation
dependence, although it restored the affinity of PN200-110 binding in
the presence of Ca
. The B
in
expressing cells was close to that in cardiac
membranes and was not altered by the coexpression of
or
(Table 1). However, B
was lower in cells expressing
both in the absence and presence of
Ca
(Table 1). That transient expression of
Ca
channel subunits in COS cells led to receptor
densities close to that in cardiac muscle demonstrates that COS cells
are suitable for studying ligand binding to Ca
channels. These results indicate that the complex formed by
is the minimum subunit
structure which qualitatively resembles native cardiac Ca
channels in terms of dihydropyridine binding.
Figure 2:
Representative Scatchard plots of
specifically bound (+)-[H]PN200-110 in
membranes from COS cells transfected with either
,
,
, or
. Only the data obtained in
the presence of 1 mM Ca
were shown. The
parameters of Scatchard analyses of binding data both in the presence
and absence of Ca
are shown in Table 1.
Figure 3:
Effects of(-)-D600 on
(+)-[H]PN200-110 binding in membrane
preparations from rabbit cardiac muscle and COS cells transfected with
Ca
channel subunits. The combinations of subunit are
illustrated in the figure. Binding assays were carried out using 0.4
nM (+)-[
H]PN200-110, in the
presence of 0.4 mM Mg
and in the absence of
added Ca
. Data are normalized against the binding in
the absence of(-)-D600. Control binding was 6.4 ± 2.1
fmol/mg of protein in COS cells with
, 74.7
± 21.2 fmol/mg of protein in
, 9.8 ± 2.2 fmol/mg of
protein in
, 76.9 ± 36.1 fmol/mg of
protein in
, and 126.8
± 23.6 fmol/mg in cardiac muscle. These values were lower than
those in Fig. 1because binding assays in the present figure
were performed in the absence of added Ca
. Data are
mean ± S.E., n = 4.
Coexpression of subunit with
markedly
attenuated the stimulatory effect of(-)-D600 (Fig. 3). At
concentrations between 3 and 100 nM,(-)-D600 had a
slight inhibitory effect on PN200-110 binding, which was similar to the
response obtained in cardiac membranes. However, at concentrations
above 1 µM,(-)-D600 stimulated PN200 binding with
the maximum stimulation of approximately 55% observed at 30
µM. In contrast, when
was coexpressed
with
,(-)-D600 partially inhibited
PN200-110 binding to COS cells in exactly the same manner as in cardiac
microsomal membrane, as illustrated in Fig. 3. Coexpression of
with
did not
cause a further change in the allosteric effect of(-)-D600. These
results once again suggest that the complex of
is the minimum combination of
subunits that resemble cardiac Ca
channels in
dihydropyridine binding.
The effect of diltiazem was tested using
similar assays in the presence of added Ca. Diltiazem
has been reported to increase, decrease, or have no effect on
dihydropyridine binding. Its effect is dependent on factors such as
temperature, divalent cations, and membrane potential. It is thus
important to define carefully the experimental conditions under which
diltiazem affects dihydropyridine binding. In the present study,
diltiazem was tested in binding assays performed at room temperature
(22-24 °C), in the presence of 0.4 mM Mg
, 1 mM Ca
, and 0.4
nM [
H]PN200-110. Under these conditions,
diltiazem had a slight inhibitory effect on PN200-110 binding to
cardiac microsomal membranes; the maximum inhibition was less than 15% (Fig. 4). However, in COS cells expressing
, diltiazem stimulated PN200-110 binding in a
concentration-dependent manner. Binding was 270% of control in the
presence of 30 µM diltiazem. This discrepancy in the
effects of diltiazem between cardiac muscle and COS cells expressing
is similar although not identical to that
observed with the effects of(-)-D600. Coexpression of
,
, or both, eliminated this discrepancy. As
shown in Fig. 4, diltiazem slightly inhibited PN200-110 binding
in COS cells transfected with
,
, or
; the
concentration-response relationships in these cells were very similar
to that in cardiac membranes.
Figure 4:
Effects of diltiazem on
(+)-[H]PN200-110 binding in membrane
preparations from rabbit cardiac muscle and COS cells transfected with
Ca
channel subunits. Subunit combination for each
curve is illustrated in the figure. Binding assays were carried out
using 0.4 nM (+)-[
H]PN200-110, in
the presence of 0.4 mM Mg
and 1 mM Ca
. Data are normalized against the binding in
the absence of diltiazem. Control binding was 23.3 ± 8.1 fmol/mg
of protein in
, 95.7 ± 26.2 fmol/mg of
protein in
, 43.0 ± 7.5
fmol/mg of protein in
, 103.3 ± 3.4
fmol/mg of protein
, and
135.2 ± 32.7 fmol/mg of protein in cardiac membranes. Data are
mean ± S.E., n = 4.
Figure 5:
Inhibition of Ca channel currents by Ca
channel antagonist
nisoldipine. Ca
channel subunits were expressed in Xenopus oocytes and currents recorded in 10 mM Ba
using the cut-open oocyte voltage clamp
technique. A, currents induced by repeated depolarization to
+10 mV from -40 mV holding potential in oocytes injected
with
and
. Nisoldipine was added in a
cumulative manner to the bath (the top chamber). The lower-case
letters beside the traces indicate currents in the absence (a) and presence of 0.1 nM (b), 10 nM (c), 1 µM (d), and 30 µM (e) nisoldipine. B, cumulative dose-response
relationship of nisoldipine in oocytes injected with
(n = 8) and
(n = 9). Data
are percentage of control currents and shown as mean ±
S.E.
Current-voltage relationships in the presence and absence of
nisoldipine are shown in Fig. 6. Nisoldipine inhibited currents
at all potentials tested. It inhibited peak currents to a greater
extent in oocytes injected with (Fig. 6B) than in oocytes injected with
(Fig. 6A). The position and
shape of the current-voltage relationships were not modified by
nisoldipine. The percentage of inhibition by nisoldipine at potentials
where inward currents can be reliably measured is plotted as a function
of membrane potential in Fig. 6C. Thus, when the
membrane was depolarized to potentials ranging from -30 to
+30 mV, currents were inhibited by approximately 70% in oocytes
injected with
and by approximately 80% in those
with
. The relative
effectiveness of nisoldipine was not affected by the amplitude of the
step pulse.
Figure 6:
Current-voltage relationships from oocytes
injected with (A) and
(B) in the absence
and presence of 30 µM nisoldipine. Currents were recorded
during voltage steps over the range of -50 to +60 mV in 10
mV increments from -40 mV holding potential. Data represent mean
± S.E. The percentages of inhibition of inward currents between
-30 to +40 mV are plotted as a function of membrane
potential (C).
Cloning and functional expression of cardiac and several
other L-type Ca channel
subunits
have firmly established that
is by itself sufficient
to form the voltage-gated ion-conducting pore and that
has the receptor sites for all major chemical classes of ligands,
such as dihydropyridines, phenylalkylamines, and
benzothiazepines(7, 13, 23, 35) . It
has also been generally accepted that the
subunit is an essential
component of Ca
channels, including the cardiac
L-type channel, since
has consistently been observed to modulate
significantly the biophysical characteristics of
(8, 15, 17, 18, 19) .
However, the functional role of
in cardiac L-type
Ca
channels has not been clearly defined, although
its presence in cardiac muscle has been demonstrated by biochemical and
molecular means(3, 36, 37) . The most
consistent effect of
has been an approximately 2-fold
increase in current density over
alone when expressed
in oocytes(13, 23) , although effects on channel
inactivation have been reported(17, 21) . Due to the
lack of measurable criteria, it is difficult to conclude whether these
effects of
are necessary for normal channel function.
In the present study, we examined the role of subunits, particularly
, on the ligand binding properties of expressed
cardiac L-type Ca
channel in two systems: transient
expression in COS.M6 cells in which dihydropyridine binding and its
allosteric regulation by Ca
, (-)-D600, and
diltiazem were studied, and expression in Xenopus oocytes
where the sensitivity of Ca
channel currents to an
antagonist nisoldipine was examined. Our results demonstrate for the
first time that
is essential for the reconstitution
of both high affinity dihydropyridine binding and its allosteric
regulation. Thus the Ca
channel complex formed by
, in comparison to that in cardiac membranes, has
several defects: 1) its affinity for PN200-110 was severalfold lower;
2) its affinity was dependent on added Ca
; and 3) its
allosteric regulation by (-)-D600 and diltiazem is abnormal.
Coexpression of
corrected all the defects associated
with
. Furthermore, consistent with the finding
that
increased the affinity of Ca
channel for antagonists, coexpression of
with
in oocytes greatly increased the potency of the
antagonist nisoldipine in blocking Ca
channel
currents. These results clearly demonstrate that
is
an essential functional component of the cardiac L-type
Ca
channel, and that
is the minimum subunit
combination of a complex that has pharmacological properties identical
to those of the native cardiac L-type Ca
channel.
Lack of
in the complex may cause conformational
changes that are not readily detected by analyzing its biophysical
properties, but that substantially alter its ligand binding ability.
In analyzing the effect of each subunit on ligand binding to
, we found that the cardiac
(
)
alone stimulated PN200-110 binding by 19-fold. The mechanism of this
stimulation was not examined in this study. By expression in Chinese
hamster ovary cells, a skeletal
subunit,
, and
another widely expressed
,
, increased the B
of PN200-110 binding to
without an effect on affinity(21, 38) . In these
studies the K
of PN200-110 was between 130 and 150
pM, similar to that in
expressing COS
cells in the present study. The increase in B
may suggest an increase in Ca
channel
expression. However, such a conclusion would be contradicted by the
observation that
did not increase the amount of
protein detected on immunoblots(22, 39) . In
contrast, Mitterdorfer et al.(22) reported that
increased 35-fold the affinity of PN200-110 to a
modified cardiac
due to a decrease in the rate the
dissociation(22) . The discrepancy may be explained by a shift
from a low affinity state (fast k
) to a high
affinity state (slow k
), if the lower affinity
state cannot be readily detected, thus leading to an apparent increase
in B
.
The present study clearly indicates
that the skeletal muscle was capable of interacting with cardiac
. In the study
showed three effects on
dihydropyridine binding to cells expressing
: it
increased binding to
by increasing affinity both
in the presence and in the absence of Ca
, it
attenuated the stimulatory effect of(-)-D600, and it completely
eliminated the stimulatory effect of diltiazem. However,
failed
to correct the divalent cation dependence of dihydropyridine binding
and it did not correct the anomalous effect of(-)-D600. The
effect of
was relatively small compared to those of
and
, and when
was coexpressed with
and
,
had no additional effect. It appears
that
is not necessary for the formation of a complex that
resembles the cardiac Ca
channel in terms of ligand
binding properties. Thus, demonstration that
interacts with the
cardiac
(or
) in this and
previous studies (17, 18) is not sufficient evidence
for the existence of a cardiac homolog of the
subunit. The
ability of
to interact with cardiac
is not
surprising given that skeletal muscle
and cardiac
share a high degree of sequence
homology(2, 13) .
The present study demonstrates
that by expressing the cloned ,
,
and
in COS cells, a Ca
channel
complex is obtained that had ligand binding affinity and allosteric
regulation identical to those in cardiac membranes. The density of
dihydropyridine receptors obtained in this transient expression system
was also close to that in cardiac membranes. Our results suggest that
transient expression of Ca
channels in COS cells
provides a useful model for studying the functional, particularly the
pharmacological, properties of Ca
channels and the
underlying structural basis for Ca
channel function.
To date six genes have been cloned as recently
reviewed by Perez-Reyes and Schneider(40) . Attempts to
correlate these cloned
subunits with endogenous
Ca
channel types have relied on the expression of
these clones followed by electrophysiological and pharmacological
characterization. Discrepancies between the potency of
-agatoxin-IVa and
-conotoxin-MVIIC to block P-type currents
in cerebellar Purkinje neurons and
-induced currents
have led to the suggestion that
does not encode the
P-type current(41) . The present study indicates a vital role
of subunit composition in determining the pharmacological properties of
a cloned channel. Numerous studies have also shown that subunit
composition can alter the biophysical properties, such as kinetics and
voltage-dependence, of cloned channels. Therefore caution must be taken
in interpreting apparent differences in the pharmacology and
electrophysiology between cloned channels and their in vivo counterparts.