(Received for publication, May 9, 1995; and in revised form, June 14, 1995)
From the
The pore-forming subunit of L-type
voltage-gated Ca
channels contains a
Ca
-binding site that is allosterically coupled to the
receptor site for dihydropyridine (DHP) Ca
antagonists. Site-directed mutations of conserved Phe and Glu
residues in the pore-lining SS1/SS2 segments greatly reduced
Ca
enhancement of DHP binding. Substitution of
Phe-1013 in the
subunit from rabbit skeletal muscle
(
) with Gly (F1013G) as in DHP-insensitive
Ca
channels caused a 4-fold decrease in sensitivity
to Ca
. Mutation of the Ca
-binding
residues Glu-1014 in domain III and Glu-1323 in domain IV to Gln
(E1014Q and E1323Q) caused 11- and 35-fold decreases in sensitivity to
Ca
, respectively, as well as decreases in the maximal
DHP binding affinities attained at optimal concentrations of
Ca
. DHP binding to the charge-reversal mutation,
E1014K, had no sensitivity to Ca
. Our results
demonstrate that high affinity Ca
binding to the Glu
residues in the SS1/SS2 segments of domains III and IV of
stabilizes the DHP receptor site in its high affinity state. We
propose a three-state model in which the affinity for DHPs is dependent
on the presence of 0, 1, or 2 bound Ca
ions at sites
in the pore.
Calcium entry through voltage-gated Ca channels is essential for regulation of excitation-contraction
coupling, excitation-secretion coupling, electrical excitability, and
other cellular functions(1) . Voltage-gated Ca
channels purified from skeletal muscle are heteropentamers
consisting of
,
,
, and
subunits (reviewed in (1) and (2) ). The
pore-forming
subunit is composed of four homologous
domains consisting of six transmembrane segments
(S1-S6)(3) . The loops that connect transmembrane
segments S5 and S6 in each domain contain the short SS1/SS2 segments
that are thought to form the ion selectivity
filter(4, 5, 6, 7, 8) .
Conserved glutamate residues in the SS2 segments (one in each domain)
form two Ca
binding sites that are essential for ion
permeation and
selectivity(4, 5, 6, 7, 8) .
The subunit also contains the high affinity
receptor site for the dihydropyridine (DHP) (
)Ca
antagonists(9, 10, 11) . DHPs act by
stabilizing the Ca
channel in different gating
states; agonists stabilize a state with high open probability, while
antagonists stabilize a state with low or null open
probability(12) . The DHP receptor site has been localized by
photoaffinity labeling and antibody mapping to peptides that correspond
to extracellular portions of the S6 segments of domains III and
IV(9, 10, 11) . In addition, mutations in the
extracellular portion of segment IVS6 affect the action of DHP
Ca
channel agonists(13) . The DHP receptor
site is thought to exist in at least two affinity states, and the high
affinity state for antagonists is stabilized by Ca
binding with a dissociation constant (K
) less than 1
µM(14) . Here, we demonstrate that high affinity
Ca
binding to the glutamate residues in the SS2
segments of domains III and IV stabilizes the DHP receptor site in its
high affinity state, and we propose a three-state model in which the
affinity for DHPs is dependent on the binding of 0, 1, or 2
Ca
ions to sites in the pore.
Experiments that determine the sensitivity of DHP
binding to cations were performed on wild-type and mutant membranes
using concentrations of (+)-[H]PN200-110
corresponding the K
of each channel for
(+)-[
H]PN200-110 determined by Scatchard
analysis in 1 mM free Ca
. Experiments
measuring Ca
sensitivity were performed in Buffer A
containing 5 mM EDTA, 5 mM HEDTA, 5 mM nitrilotriacetic acid, and various concentrations of
CaCl
. Experiments measuring Ba
sensitivity were performed in Buffer A containing 1 mM EGTA and various concentrations of BaCl
. The levels of
free Ca
and free Ba
in these
experiments were determined using published cation binding constants
for the various chelating agents(20) . Experiments measuring
Cd
sensitivity were performed in Buffer A, 100
µM CaCl
, and the indicated concentrations of
CdCl
.
Figure S1:
where K, K
, and K
are the dissociation
constants for DHP for each affinity state when 0, 1, and 2
Ca
ions are bound to the channel, and P[
], P[
Ca
], and P[
Ca
] represent
the fraction of channels in each affinity state.
The probability of
each DHP affinity state depends on whether 0, 1, or 2 Ca ions are bound. The fractional occupancy of Ca
binding sites 1 and 2 by Ca
ions is given by p
= [Ca]/([Ca] + K
The expressions for p and q above can be
substituted into to yield an expression defining DHP
binding as a function of free Ca. K
was determined empirically
as the dissociation constant for DHP binding in the presence of 1
mM Ca
. K
was assumed to be 430 nM which is the dissociation
constant for low affinity block of skeletal muscle Ca
channels by PN200-110 determined electrophysiologically (21) because two Ca
ions must bind for
permeation.
A comparison of the amino acid sequences of the SS1/SS2
segments of voltage-gated Ca channels reveals that
all DHP-sensitive channels have Phe in the SS1/SS2 segment of domain
III at the position corresponding to 1013 (Phe-1013) in
, while all DHP-insensitive channels have a Gly at
this position (Fig. 1). The adjacent Glu at position 1014 is a
component of the selectivity
filter(4, 5, 6, 7) , and this amino
acid is conserved in all voltage-gated Ca
channels.
We hypothesized that Phe-1013 might be involved in the allosteric
coupling of Ca
binding and DHP binding due to its
close proximity to the Ca
-binding Glu residue in the
pore. A mutant that replaces Phe-1013 with Gly was constructed, and
both wild-type and mutant (F1013G) constructs were transiently
expressed in tsA-201 cells. Membranes derived from these cells were
tested for the binding of the DHP antagonist
(+)-[
H]PN200-110 in the presence of various
concentrations of free Ca
. Increasing the free
Ca
causes a substantial increase in the level of
(+)-[
H]PN200-110 binding to both wild-type
and F1013G membranes. However, for the mutant channel, the EC
for this stimulation is shifted to a 4.1-fold higher free
Ca
concentration, and the amount of
(+)-[
H]PN200-110 binding at 1 nM free Ca
is decreased below the level of
detection (Fig. 2A).
Figure 1:
Sequence alignment of the P-region from
DHP-sensitive (,
,
) and DHP-insensitive (
,
,
) Ca
channels.
Amino acids corresponding to positions 1012-1016 (Domain
III) and 1321-1325 (Domain IV) of
are shown. Glu residues involved in selectivity and permeation
are indicated in bold. Phe (DHP-sensitive) and Gly
(DHP-insensitive) residues corresponding to position 1013 of domain III
are indicated with a box.
Figure 2:
Ca dependence of
dihydropyridine binding in wild-type and F1013G membranes. A,
stimulation of (+)-[
H]PN200-110 binding by
increasing free Ca
in wild-type and F1013G membranes.
Wild-type (
): EC
= 0.560 ± 0.144
µM. F1013G (
): EC
= 2.30
± 0.77 µM, p < 0.05. B and C, Scatchard transformation of equilibrium binding data at 1
mM (
), 10 µM (
), 1 µM (
), 100 nM (
), and 1 nM (
)
free Ca
in wild-type (B) and F1013G (C) membranes. D, DHP affinity,
1/K
(nM
), as
a function of free Ca
from experiments shown in B and C, above.
In order to analyze the changes in
Ca sensitivity and DHP affinity quantitatively,
Scatchard analyses were performed at various Ca
concentrations (Fig. 2, B-D). These
experiments demonstrate that 1) increasing the concentration of free
Ca
causes the DHP binding site to shift from a low
affinity state to a high affinity state in both wild-type and F1013G;
2) F1013G requires higher levels of free Ca
to
stabilize this high affinity state; 3) the K
for DHP binding to the high affinity state of mutant F1013G
is not significantly different from that of wild-type (p >
0.05); and 4) DHP binding to the low affinity state of mutant F1013G is
not detectable by radioligand binding, while wild-type has a measurable K
of 2.1 ± 0.1 nM. From
these experiments, we conclude that the high affinity state of the DHP
receptor site is not altered in F1013G, but this mutant has reduced
affinity for DHPs in the low affinity state and has a decreased
affinity for Ca
.
In general, the effects of
divalent cations on DHP binding are biphasic with enhanced binding at
intermediate concentrations followed by reduced binding as the
concentration of cation is increased(14, 22) . This
biphasic response is illustrated for Ba in Fig. 3A. The EC
for the
Ba
-dependent stimulation of DHP binding is 18-fold
higher in F1013G (EC
=109 µM) than in
wild-type (EC
= 6.0 µM), while the
IC
for Ba
-dependent inhibition of DHP
binding is not significantly different (IC
=
1-3 mM; p > 0.05). Similar results were
obtained with Co
, La
,
Mg
, Mn
, and Ni
.
These results can most easily be explained by a three-state model
describing a single DHP receptor site that exists in three
interconvertible affinity states as proposed by Glossmann et
al.(14) . Our results are most consistent with the
assumption that equilibrium among these three states is dependent on
the number of cations bound to the channel (Fig. S1).
Figure 3:
The effects of divalent cations on DHP
binding are biphasic. A, stimulation and inhibition of
(+)-[H]PN200-110 binding by increasing free
Ba
in wild-type (
) and F1013G (
)
membranes. Wild-type (
): EC
= 5.96 ±
4.86 µM, IC
= 2.67 ± 1.13
mM. F1013G (
): EC
= 108.9 ±
2.0 µM, p < 0.05; IC
= 1.1
± 0.85 mM, p > 0.05. B, inhibition
of (+)-[
H]PN200-110 binding by increasing
free Cd
in wild-type (
) and F1013G (
)
membranes. Wild-type (
): IC
= 6.58 ±
1.86 µM, n
= -2.59
± 0.71. F1013G (
): IC
= 4.18
± 0.79 µM, n
=
-2.23, p > 0.05.
K
Of the ions tested, Ca and
Cd
represented two extreme cases. The range of
Ca
concentrations that stabilized the high affinity
DHP binding site was very broad suggesting K
The results with the mutant F1013G suggest that
Ca binding to the Glu residues within the pore of the
Ca
channel may be necessary to stabilize the high
affinity state of the DHP receptor site. To test this hypothesis
directly, we altered the Ca
-binding Glu residues in
domains III and IV because photoaffinity labeling experiments suggest
that portions of these two domains are important determinants of DHP
binding(9, 10, 11) . Glu-1014 in domain III
was replaced with an uncharged Gln (E1014Q) or a positively charged Lys
(E1014K). A Lys is located at this position in voltage-gated sodium
channels(4) . Glu-1323 in domain IV was also replaced with a
Gln (E1323Q) or a Lys (E1323K), but E1323K exhibited no detectable
(+)-[
H]PN200-110 binding and was not studied
further.
Fig. 4A shows the Ca dependence of (+)-[
H]PN200-110 binding
in membranes derived from cells expressing wild-type, E1014Q, E1014K,
and E1323Q. Membranes were incubated in the presence of a constant
level of (+)-[
H]PN200-110 and the indicated
concentrations of Ca
. The concentration of
(+)-[
H]PN200-110 used was roughly equal to
the dissociation constant for high affinity binding of
(+)-[
H]PN200-110 determined by Scatchard
analysis of DHP binding at 1 mM free Ca
for
each mutant. For example, in the experiment of Fig. 4A,
the concentrations of (+)-[
H]PN200-110 used
for wild-type, E1014Q, E1014K, and E1323Q were 0.28, 1.4, 1.9, and 1.4
nM, respectively, and each gave a DHP site occupancy of
approximately 0.5 at 1 mM Ca
. The data were
fit using a mathematical model based on Fig. S1(see
``Experimental Procedures''), and the parameters determined
by this fit are summarized in Table 1. K
Figure 4:
Ca dependence of
(+)-[
H]PN200-110 binding in wild-type,
E1014Q, E1014K, and E1323Q membranes. A, membranes were
incubated with the indicated concentrations of free Ca
and (+)-[
H]PN200-110 was measured.
Data are normalized to B
and fit using a
mathematical model based on Fig. S1as described under
``Experimental Procedures.'' The results of this fit are
summarized in Table 1. B, normalized stimulation of DHP
binding from A illustrates relative shifts in EC
values for wild-type and mutants. C, inhibition of
(+)-[
H]PN200-110 binding by increasing free
Cd
. D, DHP affinity (1/K
in
nM
) determined by Scatchard analysis as a
function of free [Ca
]. For all panels:
wild-type,
; E1014Q,
; E1014K,
; and E1323Q,
.
Table 1shows that
mutation E1014Q results in a significant increase in K, while mutation E1323Q
results in significant increases in both K
and K
. This implies that, unlike
mutation F1013G, mutations E1014Q and E1323Q affect both Ca
affinity and DHP affinity. In order to study these effects in
more detail, DHP binding affinities (1/K
)
were determined from saturation binding experiments and Scatchard
analyses on wild-type, E1014Q, E1014K, and E1323Q membranes in the
presence of a range of concentrations of free Ca
(Fig. 4D). With the exception of E1014K, all
exhibited increased affinity for
(+)-[
H]PN200-110 with increasing
Ca
, although the highest affinity reached as well as
the concentration of Ca
required to reach maximum
affinity varied substantially. The maximum DHP affinities attained in 1
mM free Ca
(K
) for E1014Q, E1014K,
and E1323Q are significantly less than for wild-type (Fig. 4D and Table 1). The K
of the low affinity state (K
) measured in the presence
of 1 nM free Ca
was not significantly
different among wild-type, E1014Q, and E1014K (p > 0.05).
In contrast, E1323Q, like F1013G (Fig. 2D), has an
affinity too low to measure by radioligand binding methods at 1 nM Ca
. These results are consistent with K
values estimated from the
model of Fig. S1and the results of Fig. 4A (see Table 1) where wild-type, E1014Q, and E1014K varied only
slightly, while K
for E1323Q
was 3-fold higher than for wild-type. The simplest explanation to
account for these observations is that wild-type, E1014Q, and E1014K
can bind (+)-[
H]PN200-110 with a low but
measurable affinity without Ca
bound, while F1013G
and E1323Q have lost this ability as a result of an alteration in the
structure of the DHP receptor site or its allosteric coupling to the
Ca
binding site in the pore.
Permeation of
Ca through the pore of the Ca
channel is thought to require the binding of two Ca
ions (5, 6, 23, 24) . The
first Ca
ion binds with a K
of 0.7 µM and blocks the pore while the second
binds with a K
of about 100 mM and allows permeation(23) . Similarly, two Ba
ions are thought to bind with K
values of 16 µM(25) and 6
mM(26) , respectively. Our results indicate that Glu
residues in the pore of the Ca
channel are required
to bind one Ca
or Ba
ion to
generate the high affinity state for DHP antagonists and that the K
for Ca
binding
(K