(Received for publication, March 4, 1997, and in revised form, May 15, 1997)
From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280
The dihydropyridine Ca2+
antagonist drugs used in the therapy of cardiovacular disorders inhibit
L-type Ca2+ channels by binding to a single
high affinity site. Photoaffinity labeling and analysis of mutant
Ca2+ channels implicate the IIIS6 and IVS6 segments in high
affinity binding. The amino acid residues that are required for high
affinity binding of dihydropyridine Ca2+ channel
antagonists were probed by alanine-scanning mutagenesis of the
1C subunit, transient expression in mammalian cells, and analysis by measurements of ligand binding and block of
Ba2+ currents through expressed Ca2+ channels.
Eleven amino acid residues in transmembrane segments IIIS6 and IVS6
were identified whose mutation reduced the affinity for the
Ca2+ antagonist PN200-110 by 2-25-fold. Both amino acid
residues conserved among Ca2+ channels and those specific
to L-type Ca2+ channels were found to be
required for high affinity dihydropyridine binding. In addition,
mutation F1462A increased the affinity for the dihydropyridine
Ca2+ antagonist PN200-110 by 416-fold with no effect on the
affinity for the Ca2+ agonist Bay K8644. The residues in
transmembrane segments IIIS6 and IVS6 that are required for high
affinity binding are primarily aligned on single faces of these two
helices, supporting a "domain interface model" of dihydropyridine
binding and action in which the IIIS6 and IVS6 interact to form a high
affinity dihydropyridine receptor site on L-type
Ca2+ channels.
Voltage-gated Ca2+ channels mediate Ca2+ influx in response to membrane depolarization and thereby initiate cellular activities such as secretion, contraction, and gene expression. Several types of voltage-gated Ca2+ channels have been distinguished by their physiological and pharmacological properties and have been designated L, N, P/Q, R, and T (for review see Refs. 1-3). L-type Ca2+ channels are the molecular targets for the dihydropyridine, phenylalkylamine, and benz(othi)azepine classes of calcium channel blockers that are widely used in the therapy of cardiovascular diseases. These drugs are thought to bind to three separate but allosterically coupled receptor sites on L-type Ca2+ channels (4, 5).
The L-type Ca2+ channels consist of the
pore-forming 1 subunits of 190-250 kDa in association
with disulfide-linked
2
subunits of approximately 140 kDa, intracellular
subunits of 55-72 kDa, and, for the skeletal
muscle L-type channel, an additional transmembrane
subunit of 33 kDa (for review see Ref. 6). The
1
subunits confer the characteristic pharmacology and functional
properties of each channel type, but their function is modulated by
association with the auxiliary subunits. The pore-forming
1 subunits can be divided into two distinct families,
L-type and non-L-type, which share less than
40% amino acid identity. The L-type Ca2+
channel
1 subunit family includes skeletal muscle
(
1S) (7), cardiac/smooth muscle/neuronal
(
1C) (8-10), and endocrine/neuronal (
1D)
(11, 12) isoforms. The non-L-type
1 subunit
family also consists of at least three distinct gene products expressed primarily in neurons: N-type (
1B) (13), P/Q-type
(
1A) (14, 15), and R-type (
1E) (16).
The dihydropyridines (DHPs)1 are allosteric
modulators that act on L-type Ca2+ channels as
either agonists or antagonists (reviewed in Refs. 17 and 18). Charged
DHPs are thought to traverse an extracellular pathway to gain access to
the DHP receptor site located within the lipid bilayer 11-14 Å from
the extracellular surface of the cell membrane (19-22). Photoreactive
DHPs specifically label the 1 subunit of the
Ca2+ channel (23-29). The predominant site of labeling
corresponds to transmembrane segment IIIS6 and a portion of the
extracellular segment connecting IIIS5 and IIIS6, and approximately
15-30% of the total photolabeling is incorporated into a peptide
fragment composed primarily of transmembrane segment IVS6 (30-33). In
addition to these membrane-associated regions, a site in the
intracellular carboxyl-terminal domain has been photoaffinity-labeled
by photoreactive DHPs (34). Analysis of chimeric Ca2+
channels implicated the IIIS5 (35), IIIS6 (35), and IVS6 (35-37)
transmembrane segments in DHP binding, in general agreement with
previous photoaffinity labeling results (30, 31).
Site-directed mutagenesis of single amino acid residues that differ
between L-type and non-L-type Ca2+
channels revealed multiple amino acid residues on one side of the IIIS6
and IVS6 putative transmembrane helices that are important determinants of high affinity binding of DHP agonists and antagonists to L-type Ca2+ channels (37, 38). In addition,
mutation of a Tyr residue (Tyr1048 in
1S) in
segment IIIS6 that is conserved in all Ca2+ subtypes was
found to have the largest effect on DHP affinity (38). Mutation of this
residue to Phe caused a 12-fold reduction in binding affinity, and
mutation to Ala prevented measurable high affinity DHP binding. In the
experiments reported here, we have used alanine-scanning mutagenesis to
map both conserved and L-type-specific amino acid residues
that contribute to the high affinity of the DHP receptor site.
tsA-201 cells were provided by Dr. Robert
DuBridge (Cell Genesis, Foster City, CA). cDNA encoding the
2
subunit cloned from rabbit skeletal muscle calcium
channel was provided by Drs. Steven B. Ellis, Michael M. Harpold (Salk
Institute Biotechnology/Industrial Associates, Inc., La Jolla, CA), and
Arnold Schwartz (University of Cincinnati College of Medicine,
Cincinnati, OH). Zem 228C was provided by Dr. Eileen Mulvihill
(Zymogenetics, Seattle, WA). Full-length
1CII in Zem 229 was provided by Dr. Terry P. Snutch (University of British Columbia,
Vancouver, Canada). It encodes an isoform of
1C cloned
from rat brain (10). The expression plasmid pRc/CMV was obtained from
Invitrogen Corporation (San Diego, CA). Phage M13mp19 was obtained from
Life Technologies, Inc. (+)-[3H]PN200-110 (78-86
Ci/mmol) was purchased from NEN Life Science Products. (±)-PN200-110
was purchased from Research Biochemicals International (Natick,
MA).
For the
construction of mutant 1CII subunits (10), a
1.5-kilobase EspI fragment of the
1CII
subunit of the rat brain Ca2+ channel was subcloned into
the bacteriophage M13mp19 for recovery of single-stranded DNA template.
The resulting construct was used as a template for the generation of
single-stranded DNA for introduction of mutations into transmembrane
segment IVS6 of
1CII. These mutations were transferred
to the full-length
1CII subunit cDNA by subcloning the 272-base pair DraIII/DraIII fragment.
Mutations in transmembrane segment IIIS6 were made with a
single-stranded DNA template containing the 1.7-kilobase
AvrII/EcoRI fragment of the
1CII
cDNA. The IIIS6 mutations were transferred to the full-length
1CII construct using the 1.5-kilobase
SpcI/DraIII and the 272-base pair
DraIII/DraIII fragments in a three-way ligation.
Site-directed mutagenesis was performed using established procedures
(39), mutations were verified by sequence analysis of approximately 200 base pairs surrounding the site of mutagenesis, and the integrity of
the full-length mutant Ca2+ channel cDNA was confirmed
by extensive restriction digest analysis.
Human tsA-201
cells, a simian virus 40 (SV40) T-antigen expressing derivative of the
human embryonic kidney cell line HEK293, were maintained in Dulbecco's
modified Eagle's medium/Ham's F-12 medium (Life Technologies, Inc.)
enriched with 10% fetal bovine serum. Human tsA-201 cells were
cotransfected with wild type or mutant 1CII and cDNA
encoding the
1b (40) and
2
Ca2+ channel subunits such that the molar ratio of the
plasmids was 1:1:1. Cells were transfected by calcium phosphate
precipitation according to the procedures outlined by Margolskee
et al. (41), and cells were harvested 30-40 h following
transfection.
Cells were washed two times,
scraped, and homogenized using a glass-teflon homogenizer in Buffer A
(50 mM Tris, 100 µM phenylmethanesulfonyl fluoride, 100 µM benzamidine, 1.0 µM
pepstatin A, 1.0 µg/ml leupeptin, and 2.0 µg/ml aprotinin, pH 8.0).
The homogenate was centrifuged at 700 × g for 5 min.
The resulting pellet was discarded, and the supernatant was centrifuged
30 min at 100,000 × g. The supernatant was discarded,
and the membrane pellet was washed and homogenized in Buffer A. The
resulting membrane homogenate was divided into aliquots and stored at
80 °C for up to 3 months with no detectable loss of
(+)-[3H]PN200-110 binding activity.
Equilibrium binding assays were performed in buffer A (50 mM Tris, 100 µM phenylmethanesulfonyl fluoride, 100 µM benzamidine, 1.0 µM pepstatin A, 1.0 µg/ml leupeptin, and 2.0 µg/ml aprotinin, pH 8.0) with 20-200 µg of membrane protein, 0.01-10 nM (+)-[3H]PN200-110 and 1 mM Ca2+ at 32 °C for 180-210 min. Nonspecific binding was determined in the presence of 1 µM (±)-PN200-110, and bound and free ligand were separated by vacuum filtration over GF/C glass fiber filters. Filters were washed using ice-cold wash buffer (10 mM Tris, 1% polyethylene glycol 8000, 0.1% bovine serum albumin, 0.01% Triton X-100, pH 8.0), and bound radioactivity was detected by liquid scintillation counting. Dissociation constants (Kd) were determined using the radioligand data analysis program LIGAND. All data are means ± S.E.
ElectrophysiologyCultures of tsA-201 cells at 75%
confluence in 35-mm cell culture dishes were transfected with a total
of 4 µg of DNA consisting of an equimolar ratio of the three channel
subunit cDNAs and 0.8 µg of CD8 cDNA. After the addition of
CaPO4-DNA, cells were incubated overnight at 37 °C in
5% CO2. Twenty h after transfection, the cells were
removed from culture dishes using 2 mM EDTA in
phosphate-buffered saline and replated at low density for
electrophysiological analysis. Transfectants were recognized by
labeling with anti-CD8 antibody-coated beads (M450 CD8 Dynabeads,
Dynal, Inc.). Barium currents through L-type
Ca2+ channels were recorded using the whole cell
configuration of the patch clamp technique. 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). Voltage pulses were applied, and data were acquired using Fastlab software (Indec Systems). Linear leak and capacitance currents have
been subtracted using an on-line P/4 subtraction paradigm. (+)-PN200-110 was applied to cells by the addition of 0.2 ml of a 6×
stock to a 1-ml bath. Inhibition of Ca2+ channel current
(carried by Ba2+) during application of (+)-PN200-110 was
monitored by 100-ms depolarizations to +10 mV from a holding potential
of
60 mV applied every 10 s until block had reached steady-state
(1-3 min). The bath saline contained 150 mM Tris, 2 mM MgCl2, and 10 mM
BaCl2. The intracellular saline contained 130 mM N-methyl-D-glucamine, 10 mM EGTA, 60 mM HEPES, 2 mM MgATP,
and 1 mM MgCl2. The pH of both solutions was
adjusted to 7.3 with methanesulfonic acid. All experiments were
performed at room temperature (20-23 °C).
Transmembrane segments IIIS6 and IVS6 contain important molecular
determinants for high affinity binding of DHPs as determined by
photoaffinity labeling (Fig. 1A; Refs. 30 and
31) and by analysis of chimeric and mutant Ca2+ channels
(Fig. 1B; Refs. 35-38). Previous molecular biological studies that support this conclusion have focused almost exclusively on
amino acid residues that are present in DHP-sensitive
L-type Ca2+ channels but not in DHP-insensitive
non-L-type channels. In the present experiments,
alanine-scanning mutagenesis of single amino acid residues in
transmembrane segments IIIS6 and IVS6 of 1CII was
employed to identify all of the amino acids, both conserved and
L-type-specific, that are important determinants for DHP
binding. Most of the amino acids in IIIS6 and IVS6 of
1CII were systematically changed to Ala and coexpressed
with
2
and
1b in tsA-201 cells (Fig.
1, C and D, shaded boxes). To assess
the relative importance of Ala1157 and Ala1467,
these amino acids were changed to Pro and Ser, respectively, as found
in DHP-insensitive Ca2+ channels. Ala was chosen for these
experiments because its substitution in
helices in the core of
proteins has minimal effects on secondary structure (42). Substitution
of Ala is therefore expected to reduce the hydrophobicity and size of
the amino acid residue in each position in these putative
helices
without causing global conformational changes.
Wild type and mutant Ca2+ channels were expressed
transiently in tsA-201 cells, membrane preparations were isolated by
differential centrifugation, DHP binding was measured in filter binding
assays using the radiolabeled DHP antagonist
(+)-[3H]PN200-110, and Kd values for
high affinity DHP binding were determined by Scatchard analysis. A high
ratio of specific to nonspecific binding was observed in binding
experiments with both wild type Ca2+ channels (not shown)
and mutant channels having substantially reduced affinity (for example,
mutant N1472A; Fig. 2A). The linearity of the
Scatchard plots (Fig. 2B) indicates a single class of high affinity binding sites with a Kd of 55 pM for wild type and 470 pM for the mutant
N1472A.
Mutation of several amino acid residues in transmembrane segments IIIS6
and IVS6 to Ala had large effects on DHP binding affinity (Fig.
3). Of the mutations in IIIS6, the largest effects on
PN200-110 binding were observed when the L-type-specific
residues Ile1153, Ile1156, Met1160,
and Met1161 were changed to Ala, resulting in
Kd values that are larger than wild type by 6.2-, 17-, 3.5-, and 9.6-fold, respectively (Fig. 3A). In
addition, mutation of three residues in IIIS6 that are conserved
between L-type and non-L-type Ca2+
channels to Ala had large effects on DHP binding. The mutants F1158A
and F1159A have Kd values that are 4.5- and 4.3-fold larger than wild type, respectively. The mutant Y1152A, which corresponds to the conserved residue Tyr1048 in
1S, which we previously showed was essential for DHP
binding (38), exhibits no detectable DHP binding at concentrations of (+)-[3H]PN200-110 up to 25 nM.
Of the amino acid residues screened by alanine-scanning mutagenesis in transmembrane segment IVS6, mutations Y1463A, M1464A, and I1471A, which affect L-type-specific residues, and N1472A, which affects a conserved residue, exhibited the largest effects, with Kd values that were larger than wild type by 2.9-, 1.6-, 2.7-, and 9-fold, respectively (Fig. 3B). The comparatively small effects of mutation of residues in IVS6 compared with IIIS6 suggest that this transmembrane segment is on the periphery of the DHP receptor site and has relatively weak interactions with bound DHPs compared with IIIS6.
We were unable to detect specific DHP binding to the 1C
mutants Y1152A, Y1458A, and F1462A using concentrations of
(+)-[3H]PN200-110 up to 25 nM. To determine
if these mutants can form functional Ca2+ channels and to
measure their affinity for DHPs, these mutants were expressed and
analyzed electrophysiologically (Fig. 4). The mutant
F1458A did not express Ba2+ currents, so it is likely that
the lack of DHP binding observed with this mutant is caused by failure
to express functional Ca2+ channels. The mutants Y1152A and
F1462A both expressed Ba2+ currents but less effectively
than wild type (Fig. 4, B and C). The voltage
dependence of activation of these mutants was comparable with that of
wild type as assessed from current-voltage relationships. In contrast,
the voltage dependence of inactivation was shifted for these mutant
Ca2+ channels (Fig. 4D). For mutant Y1152A, the
voltage dependence of inactivation was shifted to more negative
membrane potentials and became significantly less steep than wild type.
For mutant F1462A, the voltage dependence of inactivation was shifted
10 mV to more positive membrane potentials. As DHP antagonists bind with higher affinity to inactivated Ca2+ channels, changes
in the extent of inactivation at the membrane potential of our
experiments could influence DHP binding and block. However, as shown
below, the changes in DHP affinity caused by these two mutations are in
the opposite directions from those expected on the basis of the changes
in the voltage dependence of inactivation so the changes in affinity
are likely to result from molecular changes in the DHP binding site
rather than from indirect allosteric effects due to the change in
inactivation gating.
The DHP antagonist PN200-110 inhibits wild type and mutant
Ca2+ channels in a concentration-dependent
manner (Fig. 4, A-C). Analysis of concentration-effect
relationships indicates that the IC50 for inhibition of
Y1152A by PN200-110 at 60 mV is approximately 25-fold higher than
wild type (Fig. 4E). Thus, Tyr1152 is indeed an
important determinant for DHP binding. This large decrease in binding
affinity coupled with low levels of expression are likely to prevent
detection of specific (+)-[3H]PN200-110 binding to this
mutant.
In contrast, the IC50 for block of mutant F1462A by
PN200-110 was decreased from 6.8 ± 1.2 nM to 16 ± 5.8 pM, indicating that this mutation results in an
1 subunit with an affinity for PN200-110 at
60 mV that
is at least 400-fold higher than wild type (Fig. 4, B and
E). The Ba2+ currents for this mutant are very
small and fewer cells express detectable Ba2+ current.
Therefore, the failure to detect (+)-[3H]PN200-110
binding to F1462A is likely due to a combination of low expression
levels and expression in fewer cells rather than a reduction in DHP
affinity. Surprisingly, the effect of the DHP agonist Bay K8644 to
enhance Ca2+ currents was not altered in this mutant
(EC50 = 151 ± 84 pM for wild type;
EC50 = 111 ± 45 pM for F1462A,
n = 3). Thus, mutation at this position has a large and
specific effect on high affinity binding of a DHP antagonist without
effect on agonist binding and action. Analysis of additional mutations
at this position will be of interest to further define the structural
basis for the specific increase in affinity for DHP antagonists.
A variety of techniques have been used to localize
the DHP receptor site. Results from experiments using charged DHPs,
photoreactive DHPs, chimeric Ca2+ channels, and mutations
of L-type-specific amino acid residues all indicate that
the core of the DHP receptor site is formed primarily by amino acid
residues in IIIS6 and IVS6 (Fig. 1, A and B). In
this study, most of the amino acids in IIIS6 and IVS6 of
1C were systematically changed to Ala, and intrinsic Ala
residues were changed to the amino acids present in corresponding
positions in non-L-type Ca2+ channels (see Fig.
1, C and D, shaded boxes). The amino
acid residues found by alanine-scanning mutagenesis to be important for
DHP binding are indicated with circles in the second
lines of panels C and D of Fig. 1, and they
are compared with those identified by mutations of
L-type-specific residues in our previous studies in the
top lines of panels C and D in Fig. 1
(38). Our results using alanine-scanning mutagenesis have identified
four new conserved amino acid residues and two new
L-type-specific amino acid residues within transmembrane
segments IIIS6 and IVS6 whose mutation has significant effects on DHP
binding affinity. The positions of these critical amino acid residues
within the IIIS6 and IVS6
helices are illustrated in Fig.
5 where black residues indicate a reduction of DHP
binding affinity by 5-fold or more and shaded residues indicate a
significant reduction in binding affinity that is less than 5-fold by
mutation to Ala.
In segment IIIS6, the largest effects were observed when amino acid residues Tyr1152, Ile1153, Ile1156, and Met1161 were changed to Ala, resulting in Kd values that are larger than wild type by 25-, 6.2-, 17-, and 9.6-fold, respectively (Fig. 5, black residues). The mutants Phe1158, Phe1159, and Met1160 had Kd values for (+)-[3H]PN200-110 binding that were 3.5-4.5-fold higher than wild type (Fig. 5, shaded residues). The COOH-terminal portion of IIIS6 including residues Phe1158, Phe1159, Met1160, and Met1161 was not analyzed in our previous studies; therefore, all of these amino acids are newly identified residues that are important determinants of high affinity DHP binding in transmembrane segment IIIS6.
In segment IVS6, mutations of residues Tyr1463, Met1464, Ile1471, and Asn1472 had the largest effects, with increases in Kd values for (+)-[3H]PN200-110 of 2.9-, 1.6-, 2.7-, and 9-fold, respectively. In addition, mutation F1462A caused a large increase in affinity for a DHP antagonist with no change in affinity for the DHP agonist Bay K8644. In our previous study, all mutations of L-type-specific amino acid residues had similar effects on the binding affinity for DHP agonists and antagonists (38). Thus, Phe1462 may have unique interactions with DHP antagonists that both determine their affinity and differentiate between agonists and antagonist drugs.
Asn1472 in transmembrane segment IVS6 is conserved between Ca2+ and Na+ channels. When this residue is changed to Ala in Na+ channels (N1769A), the resulting mutant has a 15-fold increased affinity for local anesthetic binding to the resting state of the channel (43). It was hypothesized that this mutation creates a local conformation of the drug receptor site in the resting state of the channel which resembles the high affinity local conformation of that receptor site in the inactivated state (43). Because the affinity for drug binding is highest in the inactivated states for both Na+ and Ca2+ channels, the increase in affinity for block of resting calcium channels by DHPs observed with the F1462A mutation in this work may have a similar molecular basis as the state-dependent increase in affinity for local anesthetics caused by mutation Asn1769 in segment IVS6 of Na+ channels.
The DHP Receptor Site Involves Both Conserved and L-type-specific Amino Acid ResiduesThe largest effects on DHP binding affinity were caused by alteration of conserved amino acid residues. The largest decrease in affinity (25-fold) was seen for mutation of Tyr1152 in IIIS6. Mutation of the conserved residue Asn1472 in IVS6 also caused a major (9-fold) decrease in DHP binding affinity. Alteration of a third conserved residue, Phe1462, resulted in a 416-fold decrease in IC50 for block by PN200-110, consistent with greatly increased DHP affinity of resting Ca2+ channels. These results suggest that DHP binding involves conserved amino acid residues that are important for common aspects of the structure and function of all voltage-gated Ca2+ channels. These conserved residues are likely to be largely responsible for the low affinity binding of DHPs found in non-L-type Ca2+ channels (44).
We have also identified several L-type-specific amino acid residues in IIIS6 and IVS6 that are critical molecular determinants for DHP binding. Alteration of four L-type-specific residues in IIIS6 (Ile1153, Ile1156, Met1160, and Met1161) and three L-type-specific residues in IVS6 (Tyr1463, Met1464, and Ile1471) resulted in channels with significantly increased Kd values for (+)-[3H]PN200-110 binding. It is likely that these residues are largely responsible for the high affinity of L-type Ca2+ channels for DHPs and for the requirement of the IIIS6 and IVS6 transmembrane segments for the transfer of high affinity DHP binding to non-L-type Ca2+ channels as demonstrated in experiments with channel chimeras (35-38). Recent studies also implicate two additional L-type-specific amino acid residues in transmembrane segment IIIS5 in high affinity DHP binding and in the transfer of high affinity DHP binding to non-L-type Ca2+ channels (45).
The Domain Interface Model of Ca2+ Agonist and Antagonist BindingAll three classes of Ca2+
antagonist drugs and the DHP Ca2+ agonist drugs are
allosteric ligands that exhibit state-dependent binding and
are influenced in a reciprocal manner by binding of the other classes
of drugs to distinct but interacting sites. Their effects on
Ca2+ channel function and their interactions with each
other are similar to the actions of allosteric effectors on enzymes. In
most well studied cases, binding of allosteric effectors to enzymes is
thought to take place at subunit interfaces (46-48). Similarly, strong evidence now indicates that the agonists of pentameric nicotinic acetylcholine receptors bind at sites that are formed at the interfaces of the subunits with the
and
subunits (49). Thus, there is
increasingly strong precedent for binding of allosteric ligands at
structural interfaces, possibly because the structure of multimeric proteins is most flexible in those regions. Our results indicate that
the core of the receptor site for DHP antagonists is shared between
transmembrane segments IIIS6 and IVS6, in agreement with the
predictions from photoaffinity labeling studies (30-32). Amino acid
residues in analogous positions in segments IIIS6 and IVS6 appear to
contribute to high affinity binding of DHPs (Fig. 5). Our results,
therefore, are consistent with a "domain interface model" for DHP
action proposed previously (32). We hypothesize that DHP binding at the
interface between domains III and IV influences domain-domain
interactions that are important for channel gating. Interdomain
interactions have been observed for voltage-dependent activation of K+ channels formed from four identical
subunits (50). Such interactions may also take place among the four
different domains of Na+ and Ca2+ channels
during the voltage-dependent activation process.
Site-directed mutagenesis experiments demonstrate that Ca2+ binding in the pore of the Ca2+ channel is allosterically coupled to DHP binding (51, 52). In the context of the domain interface model, DHP binding between transmembrane segments IIIS6 and IVS6 may cause conformational changes that are transmitted to the pore region of the channel resulting in increased affinity for Ca2+. We have postulated that this increased affinity for Ca2+ stabilizes a blocked state of the channel having Ca2+ tightly bound in the pore (51).
The domain interface model of drug action can now be generalized to the phenylalkylamine and benz(othi)azepine receptor sites as well as the DHP receptor site based on recent studies (37, 38, 53-55). All three classes of Ca2+ antagonist drugs interact with receptor sites formed by the IIIS6 and IVS6 transmembrane segments and are therefore well positioned to alter protein-protein interactions at the interface between domains III and IV. Thus, the modulation of Ca2+ channel function by all three classes Ca2+ antagonist drugs appears to be structurally analogous to the regulation of allosteric enzymes by their effectors.
Allosteric Interactions among Dihydropyridines, Phenylalkylamines, and Benz(othi)azepinesLigand binding studies indicate that the phenylalkylamines, benz(othi)azepines, and DHPs bind to three separate receptor sites that interact allosterically (4, 5). This requires that all three classes of drugs be able to occupy their receptor sites simultaneously. Consistent with the existence of separate receptor sites for phenylalkylamines versus DHPs and benz(othi)azepines, permanently charged DHPs and benz(othi)azepines approach their receptor sites from the extracellular side, whereas charged phenylalkylamines approach their receptor site from the intracellular side (19-22, 56-58). The close proximity of the receptor sites for DHPs, phenylalkylamines, and benz(othi)azepines (37, 38, 53-55) raises the possibility that individual amino acid residues may be required for high affinity binding of more than one of these ligands. In agreement with that expectation, amino acid residues required for high affinity binding of two or more classes of these drugs have been identified (e.g. Tyr1463, Ala1467, and Ile1471; Refs. 37, 38, and 53-55 and this study). In fact, Tyr1463 is essential for high affinity binding of all three classes of Ca2+ channel antagonists. How can these findings be accommodated with earlier results indicating separate binding sites for phenylalkylamines, benz(othi)azepines, and DHPs? Phenylalkylamines are considered to block Ca2+ channels directly by occluding the transmembrane pore through which Ca2+ ions move. In contrast, DHPs are thought to bind to identical or overlapping sites at which agonists increase Ca2+ channel activity and antagonists reduce Ca2+ channel activity, so they cannot bind in a manner that blocks the pore. Therefore, it is our working hypothesis that these different classes of drugs bind to closely spaced sites that have both shared and distinct molecular components and have different spatial relationships to the pore. Binding of DHPs affects pore function allosterically, whereas binding of phenylalkylamines blocks the pore directly. The relationships among the amino acid residues that comprise these receptor sites are defined in more detail in the following paper (59).