(Received for publication, March 14, 1997, and in revised form, May 15, 1997)
From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280
Recent studies of the phenylalkylamine binding
site in the 1C subunit of L-type
Ca2+ channels have revealed three amino acid residues in
transmembrane segment IVS6 that are critical for high affinity block
and are unique to L-type channels. We have extended this
analysis of the phenylalkylamine binding site to amino acid residues in
transmembrane segment IIIS6 and the pore region. Twenty-two consecutive
amino acid residues in segment IIIS6 were mutated to alanine and the conserved Glu residues in the pore region of each homologous domain were mutated to Gln. Mutant channels were expressed in tsA-201 cells
along with the
1b and
2
auxiliary
subunits. Assay for block of Ba2+ current by (
)-D888 at
60 mV revealed that mutation of five amino acid residues in segment
IIIS6 and the pore region that are conserved between L-type
and non-L-type channels (Tyr1152,
Phe1164, Val1165, Glu1118, and
Glu1419) and one L-type-specific amino acid
(Ile1153) decreased affinity for (
)-D888 from
10-20-fold. Combination of the four mutations in segment IIIS6
increased the IC50 for block by (
)-D888 to approximately
9 µM, similar to the affinity of non-L-type
Ca2+ channels for this drug. These results indicate that
there are important determinants of phenylalkylamine binding in both
the S6 segments and the pore regions of domains III and IV, some of which are conserved across the different classes of voltage-gated Ca2+ channels. A model of the phenylalkylamine receptor
site at the interface between domains III and IV of the
1 subunit is presented.
L-type Ca2+ channels are found in many
excitable cell types, including muscle, neuronal, and endocrine cells,
where they initiate Ca2+-dependent responses
such as contraction and secretion (reviewed in Refs. 1 and 2). The pore
forming 1 subunits of voltage-gated Ca2+
channels consist of four homologous domains (I-IV), each containing six
putative transmembrane segments (S1-S6) (3). The role of L-type channels in initiating muscle contraction within the
cardiovascular system has made them important therapeutic targets for
the treatment of hypertension, angina pectoris, and cardiac arrhythmia
(4). Three major classes of L-type Ca2+ channel
blockers are currently in clinical use, dihydropyridines, benz(othi)azepines, and phenylalkylamines. Drugs from all three classes bind to the pore-forming
1 subunit of
L-type Ca2+ channels in a manner that suggests
that their binding sites are closely linked (5, 6). Recently, much
progress has been made toward the characterization of Ca2+
antagonist receptor sites at the molecular level (for review see Ref.
7). Photoaffinity labeling studies and studies utilizing chimeric and
mutant channels have indicated that both transmembrane segments IIIS6
and IVS6 are involved in forming the dihydropyridine (8-10) and
benz(othi)azepine (11) binding sites, and that dihydropyridine binding
involves transmembrane segment IIIS5 as well (12).
In contrast, such studies of the phenylalkylamine binding site, to
date, have indicated the involvement of only transmembrane segment
IVS6. The high affinity photolabel LU49888 was found to derivatize
transmembrane segment IVS6 of the skeletal muscle L-type channel exclusively (13). In subsequent studies with mutant channels,
three amino acid residues in IVS6, which are unique to
L-type channels, have been found to be critical for high
affinity phenylalkylamine block (14, 15). Simultaneous mutation of these three residues, Tyr1463, Ala1467, and
Ile1470, resulted in channels that were blocked by the high
affinity phenylalkylamine ()-D888 with an affinity similar to that of non-L-type channels. Transfer of the L-type
IVS6 sequence in chimeric Ca2+ channels was sufficient to
confer L-type sensitivity to (
)-D888 in a
non-L-type channel (16).
Despite the studies implicating only segment IVS6 in phenylalkylamine
binding, several lines of evidence suggest that IIIS6 might also be
involved in the phenylalkylamine receptor site. The allosteric
interactions among all three major classes of L-type Ca2+ channel blockers suggests that the binding sites for
these drugs are near each other but not identical. Because evidence
from both photolabeling studies and analysis of mutant channels has
implicated domain IIIS6 in the binding of both dihydropyridines and
benzothiazepines, it is important to examine the role of individual
amino acids in domain IIIS6 in block of L-type channels by
phenylalkylamines. We have individually mutated 21 amino acids in IIIS6
to Ala and another (Ala1157) to Pro and analyzed the mutant
channels for sensitivity to the high affinity phenylalkylamine
()-D888. We report here that four amino acid residues in
transmembrane domain IIIS6 are required for high affinity block of
L-type Ca2+ channels by (
)-D888.
The phenylalkylamines are thought to block ion channels by occluding
the ion-conducting pore and thereby preventing cation permeation
(17-19). Moreover, at physiological pH, phenylalkylamines are
predominantly positively charged due to protonation of the tertiary
amino group. In view of these observations, we mutated four conserved
Glu residues known to contribute to the ion-selective pore of
L-type Ca2+ channels, Glu363,
Glu709, Glu1118, and Glu1419 (20),
to Gln and screened the resulting mutant channels for sensitivity to
()-D888. We report here that Glu1118 and
Glu1419 in the putative pore-lining segments of homologous
domains III and IV, respectively, are also involved in high affinity
block of L-type Ca2+ channels by
phenylalkylamines.
All mutations were constructed
using oligonucleotide-directed mutagenesis as described previously
(21). The IIIS6, E1118Q, and E1419Q mutations were inserted into
full-length 1 subunit constructs in the expression
vector Zem229 (Dr. Eileen Mulvihill, Zymogenetics Corp., Seattle) using
the 1.5-kilobase SpeI/DraIII fragment and the
272-base pair DraIII/DraIII fragment in a
three-way ligation. The E363Q mutation was inserted into the
full-length
1 subunit construct using the 1.4-kilobase
NgoMI/BglII fragment. The E709Q mutation was
inserted into the full-length
1 subunit construct using the
1.3-kilobase SgrAI/SpeI fragment. All mutations were confirmed by cDNA sequencing.
tsA201 cells, a subclone of the human embryonic kidney cell line HEK293 that expresses SV40 T antigen (a gift of Dr. Robert Dubridge, Cell Genesis, Foster City, CA), were maintained in monolayer culture in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Life Technologies, Inc.), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), and incubated at 37 °C in 10% CO2.
ExpressionWild type and mutant 1CII channel
subunits (22) were expressed with
1b (23) and
2/
1 (24) channel subunits and CD8 antigen
(EBO-pCD-Leu2, American Type Culture Collection) in tsA-201 cells
(derived from HEK 293 cells) by transient CaPO4
transfection as described (25). Transfectants were selected by labeling
with anti-CD8 antibodies conjugated to latex beads (Dynal A.S., Oslo, Norway).
()-D888 was applied to cells recorded
in the whole cell patch-clamp configuration by the addition of 0.2 ml
of a 6× stock to a 1-ml bath. The extracellular (bath) saline
contained 150 mM Tris, 4 mM MgCl2,
10 mM BaCl2, and pH adjusted to 7.3 with methanesulfonic acid. Patch electrode saline (intracellular) contained 130 mM N-methyl-D-glucamine, 10 mM EGTA, 60 mM HEPES, 2 mM MgATP, 1 mM MgCl2, and pH adjusted to 7.3 with
methanesulfonic acid. All experiments were performed at room
temperature (20-23 °C). No nonlinear outward currents were detected
under these conditions. Patch electrodes were pulled from VWR
micropipettes and fire-polished to produce an inner tip diameter of
4-6 µm. Currents were recorded using a List EPC-7 patch clamp
amplifier and filtered at 2 kHz (8-pole Bessel filter,
3 db). Data
were acquired using Basic-Fastlab software (Indec Systems).
Voltage-dependent currents have been corrected for leak
using an on-line P/4 subtraction paradigm.
The
L-type Ca2+ channel 1C subunit
(22) was expressed in tsA-201 cells (25) together with the
1b (23) and
2
1 (24) subunits. Ba2+ currents through the resulting
L-type Ca2+ channels were blocked by (
)-D888;
a concentration of 50 nM (
)-D888 reduced the
Ba2+ current by approximately 50% (Fig.
1A). The block by (
)-D888 was rapid and
reached equilibrium within 200 s (14). Analysis of equilibrium
block of Ba2+ currents by a range of concentrations of
(
)-D888 yielded an IC50 of 48 ± 5 nM
(Fig. 1F).
Effects of Mutations in Transmembrane Segment IIIS6 of the
The putative transmembrane segment IIIS6
of the 1C subunit of L-type Ca2+
channels contains primarily hydrophobic amino acid residues.
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All of the mutant 1 subunits studied formed functional
Ca2+ channels in tsA-201 cells except A1157P. Seventeen of
these mutations had no effect on the concentration dependence of block
by (
)-D888, as illustrated for M1160A in Fig. 1 (B and
F). In contrast, three of these single amino acid mutations
caused marked increases in the IC50 values for block of
Ba2+ currents by (
)-D888. For example, the mutation
F1164A caused a large increase in the concentration of (
)-D888
required for block of the Ba2+ current (Fig. 1C)
and approximately a 10-fold shift to higher concentration of the
inhibition curve for (
)-D888 (Fig. 1F). The
IC50 values for block of Ba2+ currents through
the wild type and all of the mutant
1C subunits are
illustrated as a bar graph in Fig. 2. Of the 21 amino
acid mutations to Ala studied, only I1153A (IC50 = 601 ± 106 nM), F1164A (IC50 = 449 ± 83 nM), and V1165A (IC50 = 449 ± 93 nM) caused significant increases in the IC50
for (
)-D888 block (Fig. 2).
In contrast to the other mutations studied, Y1152A reduced the
IC50 for ()-D888 approximately 7-fold to 7.5 ± 2.4 nM, suggesting a potentially important role of the amino
acid in this position in determining affinity for phenylalkylamines. To
assess the effect of a more conservative substitution at this postion,
we mutated Tyr1152 to Phe instead of Ala. The Y1152F
mutation, which removes only a hydroxyl group, caused an increase in
the concentration of (
)-D888 required for block of Ba2+
currents (Fig. 1D) and approximately an 18-fold shift to
higher concentration in the inhibition curve for (
)-D888 (Fig.
1F; IC50 = 873 ± 219 nM).
Thus, our results suggest that a total of four amino acid residues in
segment IIIS6 may contribute to formation of the high affinity receptor
site for (
)-D888 (Fig. 2).
If these four amino acid residues are determinants of high
affinity binding of ()-D888, mutation of combinations of them should
increase the IC50 for block of the L-type
Ca2+ channels to a value near 5 µM, like the
N-type Ca2+ channels (14). The quadruple mutation
Y1152F,I1153A, F1164A,V1165A (YIFV) caused an even further increase
in the IC50 for block by (
)-D888 than the single
mutations. Concentrations of (
)-D888 in the range of 5 µM were required to observe significant block of
Ba2+ currents (Fig. 1E). Analysis of the
block of Ba2+ currents by a range of concentrations of
(
)-D888 at equilibrium yielded an IC50 of 8.7 ± 4.8 µM (Fig. 1F).
Each concentration of drug tested in these experiments reached an
equilibrium level of block with mutant YIFV and with the other mutants
studied, indicating that the changes in IC50 reflect changes in the equilibrium Kd for drug binding. The
changes in free energy of binding of ()-D888 caused by each mutation (
[
G]) can be estimated from the measured Kd
values according to the equation:
[
G] =
RTln[Kd(wt)/Kd(mut)]. For the
single mutants with significant effects on (
)-D888 binding, the
[
G] values were: Y1152F, 1.7 kcal/mol; I1153A, 1.5 kcal/mol; F1164A, 1.3 kcal/mol; and V1165A, 1.3 kcal/mol. For the YIFV mutation, the
[
G] value was 3.1 kcal/mol. Therefore, the decrease in
binding energy in the YIFV was only 53% of the sum of the changes
caused by the individual mutations, suggesting that the contributions to the binding energy by these four amino acid residues are not independent.
To examine the
specificity of the effects of the IIIS6 mutations on Ca2+
channel function, we compared their kinetic and
voltage-dependent properties with those of Ca2+
channels containing wild type 1C. Current-voltage
relationships were generally similar for wild type and for the mutant
1C subunits having altered affinity for (
)-D888 (Fig.
3, A and B) with peak Ba2+ currents observed at +10 to +20 mV in each case.
However, closer analysis of conductance-voltage relationships revealed
small but significant differences (wild type, V1/2 = +11.5 mV; Y1152F, V1/2 = +6 mV; I1153A,
V1/2 = +3.7 mV; F1164A, V1/2 = +6.8 mV; and V1165A, V1/2 = +4.0 mV). These voltage
shifts were not due to differences in the time from forming the whole
cell patch clamp configuration because current-voltage relations were
first measured at 5 min after break-in, and no additional shifts were
observed after that time. Although the voltage dependence of activation
was significantly different for the single mutations, the voltage
dependence of channel activation was not significantly different in the
combination mutant YIFV compared with wild type (YIFV,
V1/2 = +14.3 mV), suggesting that the effects of the
single mutations were compensated in the combined mutant.
The IIIS6 mutations also affected the apparent reversal potential (Erev) of the mutant channels, which is a measure of their ion selectivity. Although the Erev of the mutant YIFV was shifted only slightly from wild type (YIFV, Erev = +56.3 ± 2.9 mV, n = 6; wild type, Erev = +61.3 mV ± 4.4, n = 10), both single Tyr1152 mutations were shifted approximately 15 mV (Y1152A, Erev = +46.9 ± 6.1 mV, n = 4; Y1152F, Erev = +46.9 ± 3.1 mV, n = 4). The Erev values of mutants I1153A and V1165A were also substantially shifted from wild type (I1153A, Erev = 47.4 ± 1.8 mV, n = 5; V1156A, Erev = 42.2 ± 2.3 mV, n = 5), whereas the Erev of mutant F1164A was more moderately affected (F1164A, Erev = 53.1 ± 3.6 mV, n = 5). These results are consistent with the idea that the IIIS6 segment contributes to the lining of the pore of Ca2+ channels, and mutations of amino acid residues in this segment therefore alter the selectivity of ion conductance.
In contrast to their lack of effect on channel activation,
phenylalkylamines cause Ca2+ channel inactivation curves to
shift in the hyperpolarizing direction, indicating that block by these
compounds is more potent at depolarized potentials where inactivation
is favored (27-29). Therefore, mutations that alter
voltage-dependent inactivation of Ca2+ channels
may affect binding and block by phenylalkylamines indirectly. To avoid
such effects, all of our experiments were carried out at a holding
potential of 60 mV, considerably more negative than steady-state
inactivation of L-type Ca2+ channels, so
further reduction in voltage-dependent inactivation by
mutations should not significantly reduce affinity for (
)-D888. Nevertheless, we have characterized the voltage dependence of inactivation of the mutant Ca2+ channels in detail (Fig.
3C). Mutation of residues Ile1153,
Phe1164, and Val1165 caused positive shifts in
V1/2 for inactivation (V1165A,
V1/2 =
14.5 mV; I1153A, V1/2 =
11.6 mV; and F1164A, V1/2 =
9.1 mV) compared with wild type (V1/2 =
17.7 mV). Removal of the
hydroxyl group from Tyr1152 did not affect the voltage
dependence of steady-state inactivation significantly (Y1152F,
V1/2 =
18.8 mV), but removal of the aromatic ring
from that position in IIIS6 resulted in a negative shift of
approximately 11 mV in V1/2 (Y1152A,
V1/2 =
29.0 mV). Inactivation curves for the
combination mutant YIFV are approximately 12 mV more positive than wild
type (V1/2 =
5.7 mV).
To examine the correlation between shifts in the voltage dependence of
inactivation and the affinity for block by ()-D888 quantitatively, we
plotted the IC50 values for the IIIS6 mutants against their
half-inactivation voltage (Fig. 3D). Half-inactivation values range from
29 mV to
4 mV, and no overall correlation with
IC50 is evident. For example, mutant Y1152F has nearly the same V1/2 value as wild type but substantially
increased IC50. Many other mutants have substantially
increased or decreased values of V1/2, but no change
in IC50. Mutants I1153A, V1165A, and F1164A have different
half-inactivation potentials but comparable increases in
IC50 values (Fig. 3D). Thus, because
inactivation is minimal at the holding potential used in these
experiments (
60 mV) and no correlation of IC50 with
V1/2 is observed, the decrease in (
)-D888 affinity
cannot be ascribed to changes in the intrinsic voltage dependence of
channel inactivation in the mutants. On the other hand, the correlation
plot of Fig. 3D may reveal the reason for the increase in
affinity caused by mutation Y1152A. This mutant has by far the most
negative V1/2 value (
29 mV) and also has the highest affinity for (
)-D888. Enhanced steady-state inactivation at
60 mV for this mutant may contribute substantially to its increased
affinity for (
)-D888.
The 1 subunits of voltage-gated
Ca2+ channels contain four highly conserved P-loops between
the S5 and S6 transmembrane segments of each homologous domain that
together form the selectivity filter through which the channel conducts
cations. The high selectivity of voltage-gated Ca2+
channels for divalent cations over monovalent ions and for
Ca2+ ions over other divalent ions is mediated by four Glu
residues, one in each homologous domain, that are conserved across all
Ca2+ channels (20, 30). Because phenylalkylamines are
thought to block ion channels by binding in the pore and the protonated amino group of (
)-D888 is positively charged at physiological pH and
could potentially interact with the pore Glu residues through an
electrostatic mechanism, we mutated each of these Glu residues individually to Gln to neutralize the negative charge that the acidic
side chains have at physiological pH and tested the affinity for block
by (
)-D888 in the four E
Q mutants. The mutant channels E363Q and
E709Q having mutations in domains I and II, respectively, were both
blocked by (
)-D888 with IC50 values that were not
significantly different from that of the wild type channel (Fig.
4, A and D). However, the mutant
channels E1118Q and E1419Q in domains III and IV, respectively, both
had significant increases in the IC50 for (
)-D888 block
(Fig. 4, B and D). The E1118Q mutant caused approximately a 20-fold rightward shift in the concentration dependence of block by (
)-D888 (Fig. 4D; IC50 = 949 ± 275 nM). The E1419Q mutant caused approximately a
15-fold rightward shift in the concentration dependence of block by
(
)-D888 (Fig. 4, B and D; IC50 = 717 ± 241 nM). These results indicate that the P-loop
Glu residues in domains III and IV interact with bound (
)-D888,
but those in domains I and II do not.
Functional Properties of Pore Mutations
Besides the effects
on ()-D888 block, mutation of the pore Glu residues affected other
parameters of channel function. The current-voltage curves for these
four mutants (Fig. 4C) show that the voltage dependence of
channel activation was not significantly different from wild type.
However, the Erev of the mutant E363Q was
shifted approximately 16 mV to more negative potential
(Erev = 44.7 ± 6.9 mV, n = 5), whereas the Erev of mutant E1118Q was shifted approximately 8 mV negatively (Erev = 52.9 ± 3.9 mV, n = 5) compared with wild type
(Erev = 61.3 ± 4.4 mV, n = 10). Surprisingly, Erev of the corresponding
mutations in domains II and IV, E709Q and E1419Q, were not different
from wild type (E709Q, Erev = 63.5 ± 4.5, n = 5; E1419Q, Erev = 57.5 ± 4.0, n = 6). The effects of the pore mutations on
the voltage dependence of inactivation also varied widely. In mutant
E363Q, the V1/2 for inactivation was not substantially different from wild type (E363Q V1/2 =
16.3 mV), whereas V1/2 values for inactivation of
E709Q and E1419Q were shifted to more positive potentials by
approximately 10 and 6 mV, respectively (E709Q, V1/2 =
7.1 mV; E1419Q, V1/2 =
11.6 mV). As with the
mutations in segment IIIS6, there was no correlation between the
effects of the mutations on IC50 for (
)-D888 and
V1/2 for inactivation, consistent with the
conclusion that these mutations affect the binding of (
)-D888
directly.
As summarized in Fig. 2, we have
analyzed the contribution of 21 single amino acid side chains in the
transmembrane segment IIIS6 to block by ()-D888. Of the single amino
acid residues mutated in this region, only four had significant effects
on the IC50 for (
)-D888. One of these four amino acids is
unique to L-type channels (Ile1153), whereas
the others are conserved across all types of voltage-gated Ca2+ channels (Scheme 1, underlined residues). The
involvement of primarily conserved amino acid residues in segment IIIS6
in high affinity (
)-D888 binding stands in sharp contrast to the
results of previous experiments in segment IVS6 where the only three
amino acids required for (
)-D888 binding were unique to
L-type channels (14-16). Our results show that conserved
amino acid residues play crucial roles in the action of
L-type selective drugs and demonstrate the importance of
systematic analysis of all amino acid residues in putative drug binding
sites in addition to chimeric approaches that target only
isoform-specific residues.
The single amino acid mutation in this region with the largest effect
was the conservative substitution of Phe for Tyr (Y1152F), in which
only a single hydroxyl group was removed from the native channel
structure. This result is similar to our previous observation (29) that
mutation of a Tyr in IVS6 (Tyr1463) to Phe caused a large
reduction in sensitivity to ()-D888. Simultaneous mutation of both
Tyr1152 and Tyr1463 to Phe resulted in a
channel with an approximately 100-fold increase in IC50 for
(
)-D888 and normal inactivation
properties.1 The importance of these two
hydroxyl groups, which are potential hydrogen bond donors, suggests a
privileged hydrogen bond between them and the single
meta-methoxy group, a potential hydrogen bond acceptor, on
the phenethylamine group of D888. This interaction is apparently not
accessible for the lower affinity phenylalkylamines verapamil and D600
(methoxyverapamil), which possess an additional para-methoxy
group (29).
Our results support the conclusion that the effects of the mutations of
amino acid residues Tyr1152, Ile1153,
Phe1164, and Val1165 on binding of ()-D888
results from alteration of the interactions of the side chains of these
residues with the bound drug. The effects of mutations of these
residues are highly specific; mutations of adjacent residues to Ala
have no effect on block by (
)-D888. There is no correlation between
the effects of these mutations on activation or inactivation of
Ca2+ channels and their effects on affinity for (
)-D888,
indicating that the mutations do not cause their effects by indirect
allosteric changes. Thus, our working hypothesis is that these four
amino acid residues interact with (
)-D888 when it is bound to its
receptor site on L-type Ca2+ channels with high
affinity.
As suggested previously for amino acid residues in IVS6 that are
required for high affinity phenylalkylamine block of L-type Ca2+ channels (14), it is likely that the IIIS6 amino acid
residues that affect ()-D888 block also project into the
ion-conducting pore. Like the IVS6 residue Tyr1463,
mutation of Tyr1152, Ile1153,
Phe1164, and Val1165 to Ala resulted in
significant shifts in apparent reversal potential. As we showed for
Y1463A (14), these shifts are likely to be due to increases in channel
permeability to the normally impermeant organic cation
N-methyl-D-glucamine, which is the principal
cation in the intracellular solution. Thus, mutations of
Tyr1152, Ile1153, Phe1164, and
Val1165 change the shape or size of the ion-conducting
pore.
Phenylalkylamines are thought to bind in the pore of
Ca2+ channels (17-19), and our results show that amino
acid residues in transmembrane segments IIIS6 and IVS6 that are
involved in high affinity binding of ()-D888 are also involved in
maintaining the ion selectivity of the pore (Ref. 14 and this work). We therefore investigated the effects of mutating four highly conserved Glu residues, Glu363, Glu709,
Glu1118, and Glu1419, in the pore region for
two reasons. First, these acidic amino acid side chains apparently
project into the pore to form the Ca2+ binding site(s) that
confers Ca2+ selectivity to voltage-gated Ca2+
channels (20, 30). Second, these amino acid side chains are negatively
charged at physiological pH and potentially participate in
electrostatic interactions with protonated (i.e. positively charged) phenylalkylamine molecules.
The spatial selectivity of the effects of mutations of these four pore
Glu residues is consistent with the large body of data showing domains
III and IV to be the site of binding of phenylalkylamines. Mutations of Glu1118 in domain III and Glu1419
in domain IV caused major reductions in the affinity for ()-D888, whereas mutations of Glu363 in domain I and
Glu709 in domain II had no appreciable effects. The
decreased affinity of E1118Q and E1419Q for (
)-D888 was likely not
caused by shifts in the voltage dependence of inactivation because
E709Q, the pore mutant with the largest shift in steady-state
inactivation, had no change in affinity for the drug. Although it is
possible that the decreased affinity for (
)-D888 conferred by E1118Q
and E1419Q is due to indirect effects, the specificity for these two
mutations among the four pore Glu residues and their lack of correlated effects on channel gating argue that they interact directly with the
bound drug molecule, most likely through electrostatic interactions with the positively charged amino group. This conclusion is consistent with previous studies showing that phenylalkylamines block ion channels
in their positively charged, protonated state (18, 31) and with the
evidence that phenylalkylamines bind within the pore of the
Ca2+ channel.
The results of this and previous studies (14-16) suggest that
the receptor site for ()-D888 is composed of at least four distinct subsites: IIIS6, the P-loop in the IIIS5-IIIS6 linker, IVS6, and the
P-loop in the IVS5-S6 linker. The critical role of amino acid residues
from both IIIS6 and IVS6 in (
)-D888 binding and block strongly
suggest that these two transmembrane domains are juxtaposed to form a
portion of the intracellular mouth of the ion-conducting pore (Fig.
5). Thus, our results support a "domain interface
model" of phenylalkylamine binding and block, as proposed
previously for dihydropyridines (32). The YIFV residues are arranged in two clusters of two amino acids each in IIIS6, but these four residues
do not align in precisely the same position in consecutive turns of the
helix as the YAI motif in IVS6 does (14). Nevertheless, the
deviation from strict cylindrical shape of many bundled
helices in
proteins of known structure would allow all four of these residues to
contribute to a binding site for phenylalkylamines in the pore of the
channel. We propose that the YIFV motif in IIIS6 and the YAI motif in
IVS6 act together to form a hydrophobic pocket that stabilizes
(
)-D888 bound in the pore and enhances the electrostatic interactions
between the pore Glu residues Glu1118 and
Glu1419 and the tertiary amino group of (
)-D888 (Fig.
5A). It will be of interest to determine how other
structurally related phenylalkylamines of differing affinity interact
with these components of the high affinity phenylalkylamine
receptor site.
Dihydropyridine and (
As we have shown in the preceding paper (33), mutation
of specific amino acid residues in transmembrane segments IIIS6 and IVS6 greatly reduce the affinity of the L-type
Ca2+ channel for the dihydropyridine PN200-110 as measured
by equilibrium binding. The results of mutations in IIIS6 and IVS6 on
block by ()-D888 and binding of PN200-110 are summarized in Fig.
5B. It is clear that the binding sites for these
structurally distinct molecules are intricately interwoven, because
single mutations at three residues in adjacent positions in IIIS6 and
IVS6 (Tyr1152, Ile1153, and
Tyr1463) disrupted both DHP2
binding and (
)-D888 block. However, mutations at other positions had
drug-specific effects. For example, mutation of Phe1164,
Val1165, Ala1467, and Ile1470
affected block by (
)-D888 but did not significantly affect PN200-110 binding. Conversely, mutations of five amino acids in the central region of IIIS6 (Ile1156, Phe1158,
Phe1159, Met1160, and Met1161) as
well as four amino acids in IVS6 (Phe1462,
Met1464, Ile1471, and Asn1472) each
had significant effects on DHP binding but not on (
)-D888 block. We
therefore suggest that the phenylalkylamines and DHPs bind to different
faces of the IIIS6 and IVS6 transmembrane segments and in some cases
bind to opposite sides of the same amino acid residues. In this model,
the allosteric interactions between bound phenylalkylamines and
DHPs would take place over very short distances, possibly separated by
no more than the plane of a phenyl ring or the width of an aliphatic
side chain.