Cardiac Electrophysiology Labs, Departments of Biochemistry & Molecular Biology and Medicine, the University of Chicago, Chicago,
Illinois
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Introduction |
Voltage-gated
Ca2+ channels are important pharmacological
targets for treatment of cardiovascular disease (Triggle, 1999
).
Commonly used drugs fall into three distinct classes: phenylalkylamines (PAA), benz(othi)azepines (BTZ), and dihydropyridines (DHP) (Hockerman et al., 1997b; Mitterdorfer et al., 1998). Among the most potent are
the 1,4 DHPs, including derivatives inhibiting
Ca2+ current (antagonists) and derivatives
increasing Ca2+ current (agonists). Each drug
type has separate but overlapping, or allosterically linked, binding
sites in Ca2+ channels. Many residues important
for binding of Ca2+ channel antagonists have been
identified, and substantial progress has been made in characterizing
the Ca2+ channel pore structure. If available
pore models are accurate, then they should predict a drug binding
region that can be experimentally tested.
Voltage-gated Ca2+ channels belong to a
structurally homologous superfamily of voltage-gated channels,
including K+ and Na+
channels (Hille, 2001), with four homologous domains or subunits of six
transmembrane segments S1 to S6, arranged symmetrically around a
central pore. The selectivity filter is formed by parts of the four
S5-S6 extracellular segments (P loops). The outer pore is lined by
these P loops and the inner pore is lined by four S6 segments and
possibly four S5 segments. X-ray crystallographic analysis of the
pore-forming portions of the bacterial K+
channels KcsA and MthK (Doyle et al., 1998; Jiang et al., 2002a) has
confirmed previous assumptions about structural topology of voltage-gated channels. The KcsA channel has four subunits, each with
two transmembrane segments, M1 and M2, forming an "inverted teepee". P loops formed from the K+ channel
M1-M2 connecting segment compose the 3-Å diameter selectivity region,
which is lined by main chain carbonyls of the TVGYG "signature sequence". The Ca2+ channel selectivity region
must be different, because P loop side chains play crucial roles in
determining the channel selectivity. Ca2+ channel
P loops contain a highly conserved pattern of four glutamates (the EEEE
locus), forming the selectivity region (Heinemann et al., 1992; Yang et
al., 1993; Stea et al., 1994).
In our Ca2+ channel outer vestibule model
(Lipkind and Fozzard, 2001), we used the crystal structure of the KcsA
channel, with adaptation of the P loops and the selectivity filter to
accommodate the roles of side chains in the selectivity process. This
procedure located the glutamate selectivity ring at the level of Met-96 of the KcsA M2 helices. It is likely that the KcsA structure is of a
closed channel (Jiang et al., 2002b). A major feature of drug binding
to the L-type Ca2+ channel is the dramatic
increase in drug affinity upon activation/depolarization. The structure
of an open Ca2+-activated
K+ channel (MthK) is almost identical, but it
shows a hinged bend in the M2 helix (Jiang et al., 2002a). However, the
critical glycine residues forming the hinge are missing from the L-type
Ca2+ channel domains III and IV S6 helices, so
that modeling of the Ca2+ channel's open state
is not feasible at this time.
Binding sites for all three classes of drugs are located below the
channel's selectivity filter. Indeed, quaternary amine derivatives of
PAA (verapamil) gain access to their binding site from the cytoplasm
(Hockerman et al., 1997b) and seem to inhibit the central pore by
physical occupancy. PAAs block the binding of both DHPs and BTZs,
reflecting the likely proximity of their individual binding sites
(Hockerman et al., 1997b; Mitterdorfer et al., 1998). Experiments with
block by derivatives of amlodipine, where the outer group of the DHP
ring was connected through alkyl spacers to a permanently charged head
group, have also located the DHP binding site deep inside the channel,
about 14 Å from the extracellular surface (Bangalore et al., 1994).
For reconstruction of the DHP and PAA binding sites, we followed the
methodology that we used for modeling the outer vestibule of the
Ca2+ channel, populating the KcsA M1 and M2
backbones with side chains of the respective L-type
Ca2+ channel S5 and S6 amino acid sequences
(Lipkind and Fozzard, 2001). The interface between the S6 
-helices
of domains III and IV, restricted above by P loop and selectivity
filter residues, can form a binding site outside of the central pore
for both antagonists and agonists of the DHP family. Critical
hydrophobic interactions of the antagonist port side ester group with
the helical IIIS6/IVS6 crossover residues stabilize channel closure,
and these interactions are absent for agonists. PAAs in this site
partially occlude both the DHP binding site and the central pore and
block Ca2+ channels by direct interaction with
the selectivity filter glutamates.
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Materials and Methods |
Modeling was accomplished in the Insight and Discover graphical
environment (MSI, Inc., San Diego, CA), as described previously (Lipkind and Fozzard, 2000, 2001). Molecular mechanics energetic calculations used the consistent valence force field approximation. For
minimization procedures, the steepest descents and conjugate gradients
were used.
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Results and Discussion |
Residues Identified as Important for Drug Binding.
Different
experimental approaches (photoaffinity labeling, construction of
chimeric channels between L-type and non-L-type Ca2+ channels) suggest that the two S6
transmembrane segments of domains III and IV interact with the
antagonists (Hockerman et al., 1997b; Mitterdorfer et al., 1998). In
addition, the intermediate part of the domain III S5 also contributes
to antagonist binding (Grabner et al., 1996). Alanine-scanning
mutagenesis has identified specific amino acid residues that interact
directly with each class of drugs. The data from alanine-scanning
mutagenesis of III S6 and IV S6 of Cav1.2 for
binding of (+)-[3H]isradipine (PN 200-110)
(Peterson et al., 1996, 1997) are presented in Table
1. Ten amino acid residues were
identified whose mutations reduced the affinity for PN 200-110 by 2- to
25-fold. Four amino acid residues of the domain IV S6
Tyr-1463,
Met-1464, Ile-1471, and Asn-1472have been found to be crucial for
binding of DHPs. Using a similar approach, Schuster et al. (1996)
distinguished only three domain IV S6 amino acidsTyr-1463, Met-1464,
and Ile-1471; simultaneous mutation of these three residues reduced
isradipine affinity by >100-fold. The largest effects of mutations in
IIIS6 were seen in positions Tyr-1152, Ile-1153, Ile-1156, Phe-1159, and Met-1161, with affinity decrease of 5- to 25-fold (Table 1), where
Tyr-1152 and Phe-1159 are conserved between DHP-sensitive and
-resistant isoforms (Peterson et al., 1997). It is remarkable that the
mutant Y1152F has a Kd value 12.4-fold
higher than that of the wild-type channel, so the phenolic hydroxyl
group of Tyr-1152 must also be important for DHP binding (Peterson et
al., 1996). These data also indicate the very local character of DHP
binding to only three successive turns of both IIIS6 and IVS6.
Moreover, the amino acid residues of IIIS6 and IVS6 that are required
for binding of isradipine are located in nearly analogous positions in
the putative -helices. This suggests a domain interface model of DHP
binding, with the drugs located between two inner faces of the IIIS6
and IVS6 segments (Hockerman et al., 1997b).
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TABLE 1
Effects of alanine mutations in domains III and IV S6 segments of the
L-type Ca2+ channel on binding of isradipine
((+)-[3H]PN200-110)
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The PAA binding pocket is composed of at least seven amino acid
residues (Table 2; Hockerman et al.,
1995, 1997a), based on the alanine scanning mutagenesis of binding of
the PAA derivative desmethoxyverapamil (D888). Three amino acid
residues in segment IVS6Tyr-1463, Ala-1467, and Ile-1470are
required for high-affinity block by D888, because their mutation
reduced affinity 6- to 12-fold (Table 2). The same conclusion was
reached by Schuster et al. (1996). However, Hering et al. (1996) also
included Met-1464 as a residue contributing to high-affinity PAA
interaction. Four amino acid residues of IIIS6 that are entirely
conserved throughout L-type and non-L-type Ca2+
channels can participate in PAA bindingTyr-1152, Ile-1153, Phe-1164, and Val-1165 (Hockerman et al., 1997a). However, we note some possibly
contradictory results for mutations of Tyr-1152. Y1152F decreased the
affinity to D888 by 19-fold, but the alanine mutation in this position
increased affinity by 6.3-fold. At the same time, the two residues,
Phe-1164 and Val-1165, which are very close to the intracellular C-end
of IIIS6 and are uninvolved in DHP binding, showed a ~10-fold
decrease in affinity for D888 upon alanine substitution.
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TABLE 2
Effects of alanine mutations on domains III and IV S6 segments of the
L-type Ca2+ channel on binding of ( )-D888
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S5 and S6 Alignment.
The critical step in modeling of the
inner pore of the L-type Ca2+ channel on the
basis of the KcsA structure was to determine how the
Ca2+ channel residues are aligned on the M1 and
M2 -helical backbones. To develop our original
Ca2+ channel pore model (Lipkind and Fozzard,
2001), we hypothesized that the residues interacting with drugs within
the pore are facing the pore. Such an arrangement maximizes the
coincidence of the DHP- and PAA-sensing residues with the pore-facing
residues of KcsA. The walls of the inner pore of the KcsA channel are
formed by adjacent pairs of residues: Val-95 and Met-96, Gly-99 and
Ile-100, Phe-103 and Gly-104, and Thr-107. Doyle et al. (1998) found
that the aromatic and hydrophobic rings of the N-terminal Trp residues of M2 -helices play an important role in arrangement of the
helices within the membrane. We began our alignment of this Trp residue of KcsA with the hydrophobic Phe-1454 residue on the N-end of domain IV
S6 (Stea et al., 1994). This places Tyr-1463, so important for binding
both DHPs and PAAs, on the level of the pair Val-95 and Met-96. The
same is true for Tyr-1152 of domain IIIS6. This alignment sets four
possible assignments of all of the IIIS6 and IVS6
Cav1.2 residues (Table
3). Table 3 shows two possible
alignments of the Cav1.2 IIIS6 and IVS6 segments
with the KcsA M2. For discussion, the alignments will be referenced as
follows: alignment 1 for both segments, 1,1; alignment 2 for both
segments, 2,2; alignment 1 for IIIS6 and alignment 2 for IVS6, 1,2;
alignment 2 for IIIS6 and alignment 1 for IVS6, 2,1. If Met-96 is
chosen for location of the Tyr residues (alignment 1,1 of Table 3 for
IIIS6 and IVS6, respectively), the locations of the
Ca2+ channel residues are shown in Fig.
1, after energy minimization. This is the
alignment we used previously for modeling of the L-type Ca2+ channel (Lipkind and Fozzard, 2001). The
teepee structure of the S6 -helices leads to wide separation of the
helices near the extracellular side of the pore, opening a space
between the pore-facing residues of IIIS6 and IVS6. That additional
space permits antagonist molecules to be located in the resulting
interface crevice, as well as inside the pore. For alignment 1,1, the
side chains of both drug-sensitive Tyr-1152 and Tyr-1463 residues face the pore and are located close enough to interact simultaneously with
DHP and PAA molecules. Phe-1159 and Ala-1467 form the opposing walls of
the interface crevice. The mutation F1159A decreased binding of DHP
5-fold (Peterson et al., 1997), and substitutions of Ala-1467 decreased
significantly binding of PAA (Hockerman et al., 1995). The bottom of
the crevice is formed by close contacts between bulky hydrophobic
residues Ile-1163 of IIIS6 and Ile-1471 of IVS6. Ile-1471 participates
in the binding of isradipine (Peterson et al., 1997). The neighboring
Ile-1470 is important for binding of PAA; its substitution by Ala
reduces binding of D888 by 6-fold (Hockerman et al., 1997a). Therefore,
on the level of these isoleucines, PAAs and DHPs interact with
different sides of the same -helix of IVS6. However, only for the
alignment 1 of IVS6, when Tyr-1463 of IVS6 coincides with the location
of Met-96 of M2, do the side chains of both Ile-1470 and Ile-1471 face
the pore. In the alternative alignment 2 for IVS6, Tyr-1463 is at the
level of Val-95 of M2, and Ile-1470, corresponding in this case to
Ser-102 of KcsA, is directed outside of the pore and makes immediate
contacts with the neighboring -helix of IVS5 segment. Consequently,
in this alignment, Ile-1470 is not able to interact with PAA. Another disadvantage of alignment 2 of IVS6 is that the pore-facing residue Ile-100 of M2 is substituted by Phe-1468 of IVS6, and its bulky aromatic side chain fills the space inside the crevice, preventing binding of antagonists in the interface between domains III and IV. In
alignment 1 for IVS6, this position is occupied by the small residue of
Ala-1467 (Table 3), which is conserved in all DHP-sensitive
Ca2+ channels (Stea et al., 1994). Moreover, in
the alternative alignment 2 of IVS6, Tyr-1463, substituting for Val-95
of M2, is located in a position removed from the interface crevice.
Altogether, these considerations give preference to alignment 1 of IVS6
when its Tyr-1463 coincides with Met-96 of the KcsA M2.
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TABLE 3
Two alignments of IIIS6 and IVS6 segments of the L-type
Ca2+ channel with M2 segments of the KcsA channel
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Fig. 1.
Interface crevice. The modeled structure of the
interface crevice between S6 -helices of domains III and IV (shown
by pink ribbons) for the alignment of Tyr-1152 and Tyr-1463 of the
L-type Ca2+ channel with Met-96 of the M2 helix of the KcsA
channel (alignment 1,1 in Table 3), with the pore-facing residues shown
by space-filling images. The S5 -helix of domain III is shown by a
yellow ribbon.
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Alignment 1 for domain IIIS6 directs inside the pore not only the side
chain of Tyr-1152 of IIIS6 but also that of Ile-1156, which are both
available for drug interaction (Fig. 1). Ile-1156 is most sensitive to
interaction with DHP, because its substitution by Ala reduced
isradipine binding affinity 17-fold (Peterson et al., 1997). However,
the side chains of the two amino acid residues Ile-1153 and Met-1161,
which show moderate affinity changes with substitution by Ala (~6-
and 9-fold, Table 1), are located outside the interdomain cavity in
alignment 1 of IIIS6. From this point of view, alignment 2 of IIIS6,
where Tyr-1152 coincides with Val-95 of M2, seems to be preferable.
This results in Tyr-1152, Ile-1153, Ile-1156, and Met-1161 facing the
interface crevice between domains IIIS6 and IVS6. Therefore, a
reasonable candidate for alignment is 2,1 (i.e., alignment 2 for domain
IIIS6 and alignment 1 for domain IVS6). From this analysis we cannot
distinguish unequivocally between alignments 1,1 and 2,1; therefore,
both must be considered further.
The transmembrane segment IIIS5 may also be involved in DHP binding
(Grabner et al., 1996), even though it was not photolabeled by any of
the photoreactive DHPs. Mutational analysis has distinguished two amino
acid residues of IIIS5 that contribute to DHP binding, Thr-1039 and
Gln-1043 (Mitterdorfer et al., 1996; He et al., 1997). Both amino
acids face the same side of an -helix. Therefore, it was logical to
align these Thr-1039 and Gln-1043 with those residues of the outer M1
-helix of KcsA that could form the back wall of the interface
creviceThr-32 and Leu-36 (Table 4). In this case, the conserved Gly residue in the C terminus ends of S5
segments (see Lipkind and Fozzard, 2001), Gly-1050 of IIIS5 of
Cav1.2, coincides with the identical Gly-43 of
KcsA. This is important, because in the KcsA structure, Gly-43 is in
immediate contact with the side chain of Val-91 of M2, and substitution of this small Gly by any other amino acid except Ala would disrupt the
relationship between the helices. In the position corresponding to
Val-91 the IIIS6 segment contains Ile-1147 (Table 3). The huge
1000-fold decrease in affinity for isradipine by T1039Y mutant (He et
al., 1997) is probably an indirect effect of substitution of Thr-1039
by the bulky Tyr residue (Wappl et al., 2001). Using KcsA as the
structural template, we find that the side chain of Thr-1039 is
screened by amino acid residues of the IIIS6 -helix located closer
to the pore, consistent with an indirect effect of its mutation.
Gln-1043 forms part of the back wall of the cavity (Fig. 1), where it
could participate in interactions with DHP or PAA (Wappl et al., 2001).
Huber et al. (2000) have proposed a different alignment for the
transmembrane segments of the L-type Ca2+ channel
on the basis of sequence homology with the KcsA channel by optimizing
the coincidence of identical amino acids. However, homology by this
criterion is limited; there are only four identical residues between
IVS6 and M2, and only one between IIIS6 and M2. In that alignment, the
important residue Tyr-1152 of IIIS6 is excluded from the interface
crevice entirely, and the side chains of Tyr-1152 (IIIS6) and Tyr-1463
(IVS6) are ~27 to 30 Å apart, making it impossible for simultaneous
interaction of these two DHP-sensing residues with DHP molecules.
Moreover, Huber et al. (2000) align Phe-1046 of IIIS5 segment with
Gly-43 of KcsA M1, which is stereochemically unlikely. The wide opening
of their proposed DHP binding site accommodates the DHP molecule.
However, their modeled structure fails to include the essential outer
vestibule P loops (see below), which would interfere with their binding cavity. The absence of P loops in the model of Zhorov et al. (2001) also restricts the reliability of their conclusions.
The Interdomain (III and IV) Antagonist Binding Site and the
Selectivity Filter.
Because the P loop structure must fit between
the adjacent S6 segments, it is evident that antagonist binding sites
inside the inner pore are restricted by the amino acid residues of the P loops and the selectivity filter. Location of the P loops in the KcsA
X-ray data are not sufficient to guide their location in the
Ca2+ channel, because the selectivity filter
structures must be different. The KcsA selectivity filter is 12 Å long
and formed by the four signature sequences TVGYG (75-79 in KcsA),
whereas in the Ca2+ channel, the side chain of
only one glutamic acid from each domain participates in the selectivity
filter. In the KcsA structure the sequences Met-96 to Ile-100 (MVAGI)
of the inner M2 -helix, which were aligned with the DHP or PAA
sensing residues of Ca2+ channelsTyr-1152,
Tyr-1463, Ile-1153, Ile-1156, and Ala-1467 (Table 3)are screened from
the pore by van der Waals contacts with the side chains of neighboring
P loops (Thr-74, Thr-75). Also, it is important that Gly-99 of the M2
segment of KcsA, which is very conserved in all
K+ channels and provides immediate steric contact
between the C
-H bond of this residue and the
main chain carbonyl of Ala-73 of the P loop (Doyle et al., 1998), be
replaced by the bulky residues of Ile in Ca2+
channels (Table 3). Such a substitution is impossible in
K+ channels. Therefore, the selectivity filter of
the Ca2+ channel must be located higher, closer
to the extracellular side of the membrane. As we previously considered
for modeling of the outer vestibules of Na+ and
Ca2+ channels (Lipkind and Fozzard, 2000, 2001),
the narrowing walls of the S6 teepee restrict the location of the
vestibule frame consisting of the four -helix-turn-
-strand P
loops approximately to the level of Ile-1575 of IVS6 of the
Na+ channel or of Tyr-1463 of IVS6 of the
Ca2+ channel. In both cases, we align these
residues with Met-96 of M2, coinciding with the data of alanine
scanning mutagenesis for the Cav1.2 channel. The
first residues in domains IIIS6 and IVS6 to influence binding of
isradipine and D888 were Tyr-1463 of IVS6 and Tyr-1152 of IIIS6, both
in the 10th position from the N terminus of the -helix. S6 residues
near the Tyr residues all had no effect on block by DHP or PAA
(Hockerman et al., 1997b; Mitterdorfer et al., 1998). Presumably, dense
packing by residues of the P loops or by the selectivity filter
prevents access to the residues in the first nine positions in the S6 helices.
In support of the location of the P loops near Tyr-1463 of IVS6, that
residue is intimately connected with the selectivity and permeation
process. Its mutation to alanine alters the Ca2+
channel reversal potential by 15 mV (61 mV in the wild-type channel
and 46 mV in the Y1463A mutant). It also increases permeation by
N-methyl-D-glucamine (Hockerman et
al., 1995), suggesting that Tyr-1463 is indeed located near the
selectivity filter, perhaps contributing to its structural integrity.
The same is true for Tyr-1152; its mutation to alanine also affected
selectivity (Hockerman et al., 1997a). It is therefore necessary to
locate the four glutamic acids of the selectivity filter (the EEEE
locus), arranged for formation of the high affinity
Ca2+ binding site, at the level of Tyr-1463 and
Tyr-1152. At the same time, the P loop and selectivity filter location
must also allow the aromatic rings of both of these Tyr residues to be
accessible to DHP and PAA from below the selectivity ring.
Before consideration of the binding site for DHPs and PAAs inside the
pore, we need to note that our reconstruction of the inner pore of
Ca2+ channels does not coincide exactly with that
of the KcsA channel. The -helices of the Ca2+
channel P loops must be more distant from the pore axis than the KcsA P
loop -helices to allow room for the glutamate side chains to form
the selectivity ring. Consequently, the S5 helices are slightly
displaced outward relative to the M1 segments (Lipkind and Fozzard,
2001). Optimization of the potential energy of the assembly of P loops
and S5 and S6 helices led also to additional minimal adjustment of the
S6 helices in the outward direction (1.5-2.0 Å). Displacement of the
S6 helices is also determined by the requirements of packing of side
chains in the narrowest part of the teepee, which occurs at the
crossing of the S6 helices. In the KcsA channel, the immediate contacts
of neighboring M2 helices at this level are realized by approach of
small amino acids: Thr-107 and Ala-111 from one M2 helix to Gly-104 and
Ala-108 of M2 of the adjacent subunit (Doyle et al., 1998), whereas in our Ca2+ channel model, the corresponding
positions are substituted by amino acid residues with bulky side
chainsIle, Val, and Phe (Table 3). The locations of the S6 helices
were recalculated to allow optimal packing of these bulky side chains,
with contacts between Ile-1163 and Phe-1167 of IIIS6 and Ile-1471,
Val-1475, and Ile-1478 of IVS6.
After arrangement of the plane of the selectivity filter relative
to the pore, we examined the structure of the antagonist binding site
in the interdomain crevice formed by IIIS6, IIIS5, and IVS6 segments
according to both of the alternative alignments 1,1 and 2,1 (Table 3).
Using alignment 1,1, the binding site is shown in Fig.
2, left. On the upper level, the crevice
is restricted by the side chain of Glu-1118 and the main chain of
the neighboring Phe-1117 of the P loop of domain III, which are
surrounded by the side chains of Tyr-1152 of IIIS6 and Tyr-1463 of
IVS6. This places the side chains of the tyrosines near the top of the
binding site (Fig. 2A, left), a little lower than the glutamic acids of the selectivity filter, where they are available for interaction with
drugs inside the cavity. The bottom of the interdomain crevice is
restricted by the side chains of bulky hydrophobic residues of Ile-1163
(IIIS6) and Ile-1471 (IVS6) at the III/IV S6 crossing. The side walls
of the cavity are formed by the side chains of Phe-1159 (IIIS6) and
Ala-1467 (IVS6) with separation of ~4 to 4.5 Å. The residues
N-terminal to Glu-1118 of the P loop (Phe-1117, Thr-1116, and Ser-1115)
and the side chains of Ile-1155, Phe-1159, and Asn-1162 of domain IIIS6
and Met-1464, Phe-1468, and Asn-1472 of domain IVS6 contribute to the
back wall of this cavity. It is unlikely that the S4 voltage sensor and
its surrounding water-filled compartment are behind the binding cavity;
more likely, the cavity wall is in contact with hydrophobic regions of
the protein and/or the lipid bilayer. The alternative alignment 2,1 (shifting IIIS6; Table 3) places Tyr-1152 to coincide with Val-95 of
KcsA M2. It seems less acceptable because accessibility to the side
chain of Tyr-1152 is restricted by the main chains of residues Phe-1117 and Glu-1118 of the P loop and the side chain of Ile-1156 of IIIS6 itself (Fig. 2, right). Therefore, the next step is to consider docking
DHP and PAA into this crevice constructed with alignment 1,1 (Fig. 2,
left).
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Fig. 2.
Comparison of alternative alignments. The interface
III/IV binding site of the L-type Ca2+ channel formed by S6
-helices and restricted on the top by the side chain of residue
Glu-1118 (domain IIIP) of the selectivity filter. The structure of the
site in the case of alignment of Tyr-1152 of domain IIIS6 with Met-96
of KcsA and Tyr-1463 of domain IVS6 with Met-96 of KcsA (alignment 1,1 of Table 3) is shown in the left, whereas for alignment of Tyr-1152
with Val-95 and Tyr-1463 with Met-96 (alignment 2,1 of Table 3), it is
shown in the right.
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Conformation of the DHP for Docking in the Binding Site.
Before docking the DHP molecule, we need to consider its conformational
possibilities, which have been described comprehensively by Goldmann
and Stoltefuss (1991). The most characteristic representative of the
DHP family is nifedipine (Fig. 3A). The
DHP ring itself is nearly planar and exists in a flattened boat
conformation with the 4-aryl substituent at C4 occupying the
pseudoaxial position perpendicular to the plane of the DHP ring. In DHP
structures, the polar group inside of the 4-aryl ring (here
NO2) is maximally distant from the NH bond at the
stern of the DHP ring. If the 4-aryl ring is up, then two sides of the
DHP ring can be distinguishedthe port (left) side and the starboard
(right) side. The ester substituents of the DHP ring could adopt
various conformations. At the same time, assuming a favored coplanar
arrangement of the ester carbonyl group relative to the double bond of
the DHP ring, two conformations are possible for the ester groups on
each side: trans and cis, where the carbonyl
group is located in the trans- or cis- position relative to the DHP ring double bond. X-ray analysis shows a preference for the cis arrangement of the ester group on the port side,
whereas the ester group on the starboard side may assume either
orientation (Triggle et al., 1989). The space-filling model of
nifedipine, solved by energy minimization by the Discover module of
Insight II with the cis conformation of the ester group on
the port side is shown in Fig. 3A. In this conformation, nifedipine has
an elongated shape, with the long axis passing through the port and
starboard sides.
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Fig. 3.
Space-filled images of dihydropyridines. A,
nifedipine. B, ()-Bay K8644. C, derivative of nifedipine with an
isopropyl ester group on the port side. D, DHP with fused thiophene
ring.
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The importance of a cis arrangement of the ester group on
the port side for inhibitory biological activity of the DHP antagonists was shown by fused DHPs with an additional five-membered ring on the
port side with the double bond, located exactly in the cis
orientation relative to the double bond of the DHP ring (Goldmann and
Stoltefuss, 1991). For example, the corresponding thiophene derivative
of DHP (Adachi et al., 1988) (Fig. 4A)
has shown a higher pharmacological activity than nifedipine itself. Its
optimal space-filling image is shown in Fig. 3D. The high activity of DHP with the fused thiophene nucleus suggests that the ester group on
the port side participates minimally in formation of hydrogen bonds
with the DHP receptor site. The location of the lipophilic alkyl chain
of the five-member thiophene ring is also important, because the
derivative shown in Fig. 4B with the same alkyl substitution of the
neighboring C-2 of the ring is practically inactive (Adachi et al.,
1988).
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Fig. 4.
Chemical structure of dihydropyridines with the port
side fused thiophene rings. A, active derivative. B, nonactive
derivative (Adachi et al., 1988).
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The ester group on the port side plays a definitive role in the
inhibitory function of DHP. A change in activity is mainly associated
with an increase in the size of the aliphatic chain of the ester group
on this side. The derivative of nifedipine with the isopropyl ester
group in this position is more potent by a factor of 15 than regular
nifedipine (Towart et al., 1981). Similar changes in the size of the
ester group on the starboard side did not produce such a noticeable
effect. The increase in activity with introduction of bulky nonpolar
groups suggests that the ester group on the port side participates in
hydrophobic interactions with the channel. Because of this, we begin
with docking of the nifedipine derivative with the isopropyl ester
group on the port side (Fig. 3C).
Agonist activity is produced only by substitutions by small polar
groups in the ester group on the port side, enhancing the Ca2+ channel open probability. For example, the
typical strong agonist ()-Bay K8644 (shown in Fig. 3B as a
space-filling image) has only a nitro group on the port side, whereas
the enantiomer with the nitro group on the starboard side is still a
Ca2+ antagonist (Goldmann and Stoltefuss, 1991).
Therefore, comparison of the structures of the agonists with the
antagonists allows us to conclude that introduction of the negative
electrostatic field on the port side is important for agonist activity,
whereas antagonist activity is connected intimately with the presence of the hydrophobic aliphatic chains inside the ester group on the port
side of the DHP ring.
Evidence that agonists and antagonists bind to the same site includes
similar effect of mutations in IIIS6 and IVS6 on affinities of the
antagonist isradipine and the agonist ()-Bay K8644 (Peterson et al.,
1996). Specifically, both Tyr-1048 of IIIS6 and Tyr-1365 of IVS6 of the
1S channel (corresponding to Tyr-1152 and
Tyr-1463 of 1C) are required for high-affinity
binding of both the DHP antagonists and agonists (Peterson et al.,
1996; Schuster et al., 1996). Therefore, we expect to dock ()-Bay
K8644 into the same III/IV crevice as nifedipine. However, this plan
has an additional implication stated by Hockerman et al. (1997b): "If
DHP is bound to a single site at which agonists increase
Ca2+ current and agonists reduce
Ca2+ current, then they cannot bind in a manner
that blocks the pore".
Docking the Heterocyclic DHP Ring in the Interdomain III/IV
Cleft.
The interface crevice between domains III and IV, which we
propose as a candidate for the binding site of DHP (Fig. 2, left), is
restricted by the side chains of Tyr-1152 (IIIS6) and Tyr-1463 (IVS6)
on the top and by the bulky nonpolar side chains of Ile-1163 and
Ile-1471 on the bottom. The side chains of Phe-1159 (IIIS6) and
Ala-1467 (IVS6) form the walls of the narrow cavity, with side-to-side
separation that corresponds approximately to the width of an aromatic
ring. In the DHP structure, the two aromatic ring choices are the
heterocyclic DHP ring or the 4-aryl ring. However, if the 4-aryl
substituent approaches the interface crevice, the long axis of the DHP
ring adopts a perpendicular location relative to the pore axis, such
that the ester groups on both sides contact the inner pore walls and do
not allow the 4-aryl ring to move deeply into the cavity. In contrast,
the DHP ring can be readily accommodated deep inside the cavity
parallel to the pore axis, with its stern N-H group located close to
the IIIS5 helix. In this case, the plane of the heterocyclic ring and
its long port-starboard side axis is approximately parallel to the pore
axis, whereas the 4-aryl ring is located perpendicular and closer to
the center of the pore. The height of the proposed binding cavity
corresponds to the size of the DHP ring along its long axis.
Two alternative orientations of the DHP ring are possible:
starboard-side up/port-side down or the converse. Upon docking the
nifedipine derivative with the port side isopropyl group (Fig. 3C), we
find that the total energies of nonbonded interactions with the binding
cavity are almost identical for the two orientations. The most obvious
difference between the two orientations of the DHP ring is that in the
first one the nonpolar part of the ester group on the port side
(isopropyl) approaches the hydrophobic S6 crossing, approximately on
the level of the side chains of Ile-1163 (IIIS6) and Ile-1471 (IVS6).
However, both orientations produce effective contacts with the two
DHP-sensitive Tyr-1152 and Tyr-1463 residues (Figs.
5 and
6). Consequently, we docked the DHP
ring in both conformations and determined interaction energies for
comparison.
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Fig. 5.
DHP in the interface binding crevice. Optimal
arrangement of DHP (the nifedipine derivative with isopropyl ester
group on the port side) inside of the interface binding site between S6
-helices of domains III and IV of the L-type Ca2+
channel (left). The ester group on the port side is located in the down
orientation and adopts a cis-conformation relative to
the DHP ring. Amino acid residues surrounding DHP are shown by
space-filled images in the right. The 4-aryl ring interacts with the
side chains of Tyr-1152 and Tyr-1463, whereas the nonpolar isopropyl
group interacts with the hydrophobic side chains of Ile-1156, Ile-1163,
and Ile-1471 on the bottom. The DHP ring is located in the depth of the
crevice between the side chains of Phe-1159 and Ala-1467, parallel to
the pore axis.
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Fig. 6.
Alternative orientation of DHP in the binding
crevice. Optimal arrangement of the isopropyl derivative of nifedipine
with the port side ester in the upward orientation.
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Arrangement of the nifedipine derivative with the additional isopropyl
group on the port side located down was optimized (Fig. 5), allowing
the side chains of residues of IIIS6 and IVS6 to reorient without
movement of the main chains. The overall energy of nonbonded
interactions with the binding cavity for this conformation was 35
kcal/mol. The aromatic ring of the 4-aryl substituent was located
between the side chains of Tyr-1152 (IIIS6) and Tyr-1463 (IVS6), such
that the nitro group in the ortho position could interact directly with
the hydroxyl group of the side chain of Tyr-1152, whereas the aromatic
ring itself could participate in a perpendicular aromatic-aromatic ring
interaction with Tyr-1463 (Burley and Petsko, 1988). This proposed
interaction of the 4-aryl ring with the side chain of Tyr-1152 meets
the requirement for sensitivity of DHP binding not only to its
substitution by Ala, but also the effect of substitution of this Tyr by
Phe (Table 1) (Peterson et al., 1997). Tyr-1463 is also important for
binding of isradipine (Table 1). The calculated energy of noncovalent interaction between DHP and the side chain of Tyr-1463 was 4.5 kcal/mol.
In this port-side-down orientation, the DHP ring itself is parallel to
the side chain of Phe-1159 (IIIS6) with the interaction energy of about
4 kcal/mol, and it is located very close to Ala-1467 (IVS6),
permitting the N-H group on the stern of the DHP ring to interact with
-electron cloud of the Phe-1159 aromatic ring. However, with this
proposed orientation, neither N-H nor the carbonyl groups of the ester
groups form direct hydrogen bonds. The ester group on the starboard
side is located behind the side chains of Tyr-1152 and Tyr-1163 and
near the side chain of Met-1464 (IVS6). Substitutions of Met-1464 also
reduced affinity of DHP modestly (Hockerman et al., 1995). The
starboard ester group also interacts with the side chain of Ile-1155
with calculated energy of 2.7 kcal/mol, but substitution with Ala
failed to change the calculated interaction energy. This is an
interesting example when mutation with alanine (Peterson et al., 1997)
apparently fails to disrupt a real interaction.
The most notable distinction of this docking is the numerous nonpolar
contacts of the port side ester group with hydrophobic residues on the
bottom of the cavity. The isopropyl group of this derivative of
nidefipine interacts not only with the side chains of Ile-1163 and
Ile-1471, but also with the side chain of Ile-1156 (Fig. 5);
experimentally, the Ala substitution of Ile-1156 produced the most
remarkable decrease in isradipine affinity by 17-fold (Table 1;
Peterson et al., 1997). Such an interaction is possible only in the
cis-orientation of the port-side ester group. The energies
of nonbonded interactions of the isopropyl group with each of the three
Ile residues is at least 2.5 kcal/mol. The dominant role of nonpolar
hydrophobic interactions for DHP is in accordance with the early
suggestion that the DHP receptor site is hydrophobic, on the basis of
the lipophilic character of the molecules (Herbette et al., 1989). It
is not always possible to compare quantitatively the calculated energy
of interaction with affinity change upon replacement with alanine,
because interactions depend strongly on exact spacing and other factors
that may not be modeled. The important conclusion is that the
hydrophobic amino acid residues are located near critical components of
the DHP ring.
The port-side-up orientation also produced optimal binding (Fig. 6),
with overall energy of interaction of 33 kcal/mol, almost the same as
for port-side down. The loss of nonbonded interactions of the isopropyl
ester group with Ile-1156 and the S6 crossing residues of Ile-1163 and
Ile-1471 is compensated by strong interactions of DHP with both
Tyr-1152 and Phe-1159 (about 8 kcal/mol each). The absence of
specific interactions with nonpolar residues of the S6 crossing (see
later) is a disadvantage for this docking arrangement because
hydrophobic interactions with the port side ester are important for DHP
activity (Towart et al., 1981; Goldmann and Stoltefuss, 1991). The
port-side-up orientation also seems to overestimate interactions with
the side chains of Tyr-1152 and Phe-1159, because the experimental
effects of substitution of these aromatic residues are modest (Table
1). Finally, this orientation is probably excluded by the docking
interactions of agonists (see later). Therefore, pending more specific
experimental evidence, we proceed with analysis of the port-side-down
conformation for binding of DHP.
The proposed DHP binding site can also accommodate the DPH derivative
with a fused thiophene ring on the port side (Fig. 4) only if the
iso-C4H9 substituent of the
thiophene ring is located in its 3rd position (Fig.
7, left). The height of the proposed binding cavity between Glu-1118 of the selectivity filter on the top
and residues of the S6 crossing (Ile-1163 and Ile-1471) on the bottom
corresponds exactly to the size of this derivative, which established
the same optimal interactions with the cavity side chains as described
for the nifedipine derivative with the down orientation of the
isopropyl ester group (Fig. 5). However, transfer of the
iso-C4H9 group to the 2nd
position (Fig. 7, right) resulted in formation of prohibited van der
Waals contacts of this group with the bulky hydrophobic Ile-1163 and
Ile-1471 on the bottom, which could explain the loss of biological
activity by that molecule (Fig. 4B) (Adachi et al., 1988). The
trans orientation of the aliphatic substituent of the ester
group on the port side stereochemically coincides with location of the
iso-C4H9 group substituting
in the 2nd position of the thiophene ring (Fig. 7), again explaining
the preference of the cis orientation of the ester group for
DHP binding.
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Fig. 7.
DHPs with thiophene ring in the binding site.
Arrangement inside the binding cavity of DHPs with fused thiophene
rings (see Fig. 4), having the alkyl isobutyl substitution in the third
position (active derivative) on the left, and in the second position
(nonactive derivative) on the right. Substitution in the third position
allows the optimal arrangement, whereas substitution in the second
position leads to prohibited van der Waals contacts of the isobutyl
group with bulky side chains on the bottom of the binding cavity.
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Possible Mechanism of DHP Block.
Figures 5 and 7 present side
views of the location of the DHP antagonists in the interface crevice
between S6 helices of domains III and IV. The top view of the complex
with the four domain assembly (Fig. 8)
(Lipkind and Fozzard, 2001) demonstrates more clearly the location of
the derivative of nifedipine (shown in violet) outside of the central
pore and the Ca2+ permeation pathway. If the DHP
do not obstruct the pore, then how do they block the current? The
"teepee" arrangement of M2 in the KcsA channel structure as a model
for the S6 -helices of the Ca2+ channel
provides a separation of the helix N-terminal ends, such that the side
chains of one helix do not interact with each other; near the
C-terminal ends, there is a densely packed bundle of the four
-helices (Doyle et al., 1998). In the Ca2+
channel model, the first level at which the S6 -helices interact with each other is at residues corresponding to residues 104 and 107 of
KcsA, so that the S6 immediate contacts are between the side chains of
Val and Leu of domains I and II, Phe and Met of domains II and III, Ile
and Ile of domains III and IV, and Phe and Leu of domains IV and I
(Table 3). Voltage-activated channels seem to open by movement of the
inner parts of the S6 -helices (Liu et al., 1997). Electron
paramagnetic resonance measurements of spin-labeled residues in KcsA
suggested that opening involves a change in the diameter of the inner
pore at the C-terminal crossing of the S6 helices (Perozo et al.,
1998). In this proposed model of DHP binding in the
Ca2+ channel, the nonpolar alkyl substituents of
the port side ester interact with hydrophobic residues at the bottom of
the binding cavity that forms the crossing between IIIS6 and IVS6
helices (Fig. 5). The DHP antagonists bind with higher affinity to the inactivated (closed) state of the channel (Hockerman et al., 1997b). Both observations suggest that the hydrophobic interactions of the
port-side ester group of the DHP antagonists with the hydrophobic residues could stabilize a closed state of the L-type
Ca2+ channel by preventing conformational changes
at the S6 crossing that are required for gating.
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Fig. 8.
Location of DHP outside the central pore of the
L-type Ca2+ channel. Top view of the four-domain
organization of the Ca2+ channel with the DHP molecule
(shown by violet balls and sticks) located in the interface between
domains III and IV. The selectivity filter glutamates and a
Ca2+ ion (pink ball) are also distinguished.
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DHP Agonist Binding Site.
Docking of the agonist ()-Bay
K8644 (Fig. 3B) inside the proposed binding site was by superposition
with nifedipine in its optimal arrangement, followed by minimization of
the potential energy, as shown in Fig. 9.
The DHP and 4-aryl rings of the agonist established the same optimal
energetic interactions with the side chains of Tyr-1152 and Tyr-1463
and of the other residues of the crevice/pore as they did for binding
of antagonists (Fig. 5). With the port-side-down orientation, the
specific nitro group on the port side of the DHP ring of the agonist
(Fig. 3B) is directed into the pore. In this orientation the
NO2 does not participate in specific interactions
with the IIIS6/IVS6 interface. Indeed, its substitution by hydrogen
maintains the polarization and still results in agonist behavior
(Goldmann and Stoltefuss, 1991). With the port side up, this
nitro group would be screened from the pore by side chains of Tyr-1152,
Tyr-1463, Glu-1118, and other residues of the IIIS6 and IVS6 helices,
which could lead to a loss of agonist activity. The proposed
arrangement of ()-Bay K8644 also allowed optimal docking of another
agonist CGP 28-392, which includes the five-membered lactone ring on
the port side (Goldmann and Stoltefuss, 1991). Both of its oxygens
coincided with the NO2 group of the DHP ring of
()-Bay K8644. However, in the case of docking of (+)-Bay K8644 in the
starboard-side-down orientation, the location of the
NO2 group of the DHP ring coincided with location
of the similar NO2 group of ()-Bay K8644. Then (+)-Bay K8644 could act as an agonist. However, it is an antagonist, excluding the starboard-down orientation of DHPs inside the receptor site. With orientation of the port side of ()-Bay K8644 down, the
NO2 group faces the pore, near to the S6
crossing. The loss of hydrophobic interactions with the S6 crossing
residues, which are so important for binding of antagonists, suggests
why there is low affinity of the agonist for the inactivated state, and in turn, why it may destabilize the inactivated state and increase the
probability of channel opening (Hockerman et al., 1997b). In the
proposed arrangement of antagonists, the electronic -cloud of the
aromatic 4-aryl ring is screened by the aliphatic chain on the port
side ester group. However, with agonist binding, the -cloud of the
4-aryl ring and the port side NO2 group produce additional negative electrostatic potential inside the pore that might
also contribute to stimulation of Ca2+ permeation
(Goldmann and Stoltefuss, 1991).
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Fig. 9.
Binding of ()-Bay K8644. Model of binding of the
derivative with agonist activity inside the interface III/IV domains of
the Ca2+ channel with the nitro group on the port side of
the DHP ring facing the pore.
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DHPs and the Selectivity Filter.
Direct interactions between
the DHP molecule and the selectivity filter, its glutamic acids, and
Ca2+ ions in the selectivity filter were not
considered during the development of the binding pocket, because
mutation of the selectivity filter glutamates only moderately reduce
the affinity of isradipine binding (by ~4-fold; Peterson and
Catterall, 1995). The high-affinity binding of
Ca2+ in the pore also stimulates binding of DHPs
(Peterson and Catterall, 1995; Mitterdorfer et al., 1995). It seems
unlikely that Ca2+ directly interacts with the
lipophilic DHP. Indeed, the presence of such strong acceptors of
Ca2+ as four glutamates should preclude direct
interactions with the neutral DHPs by a competitive mechanism. Peterson
and Catterall (1995) and Hockerman et al. (1997a) consider a possible
allosteric mechanism of interaction between Ca2+
and the DHP binding site. It is entirely possible that the presence of
Ca2+ in the selectivity filter also influences
the conformational state of the binding site immediately below the
domain III glutamic acid.
Next, we considered arrangement of DHPs on the III/IV interface,
outside of the pore and the selectivity filter. The fact that BTZs,
located on the same level of the pore as DHPs (Hering et al., 1996), do
not inhibit but instead stimulate binding of DHPs supports this
proposal. Location of bound DHPs outside the pore is also compatible
with new data on block of L-type Ca2+ channels by
DHPs where the DHP moiety is attached to a charged head group through
short alkyl spacers. J. Xia and R. Kass (personal communication) have
shown: 1) No effect of test voltage on block by the DHP with a tethered
charge, presumably because the charged head group fails to enter the
electric field; and 2) DHPs display similar potency when the charged
head group was positive or negative, which excludes their interaction
with the selectivity filter. These results indicate that DHPs reach
their binding site below the selectivity filter without passing through
the filter itself. Although our model does not show this alternative
path, it does locate the binding in a space between IIIS5, IIIS6, and
IVS6, immediately below the domain III P loop -helix, suggesting
that a lipophilic path for the DHP molecules may exist behind the P loop. This conclusion is in agreement with the recent observation that
mutation of Phe-1112 (F1112A) of the P loop of domain III of
1C, which we located inside of the hydrophobic
-helical segment of this P loop (Lipkind and Fozzard, 2001), also
modulated binding of both agonist and antagonist DHPs (Yamaguchi and
Adachi-Akahane, 2002). In addition, Yamaguchi et al. (2000) report that
the alanine mutation of Ser-1115 in the IIIP, which we locate at the
top of the binding cleft, markedly reduced agonist efficacy. Therefore, the N-terminal segment of IIIP also could play an important functional role, allowing DHPs to reach the interface binding site inside the
L-type Ca2+ channel.
Docking of PAA Inside the Ca2+ Channel Pore.
PAAs
block L-type Ca2+ channels from the intracellular
side and are considered to block by occlusion of the central permeation path (Hockerman et al., 1997b). Three molecules within the PAA class
have been extensively studied: verapamil (shown in Fig. 10 by a space-filled image), D888, and
methoxyverapamil. D888 contains only one meta-methoxy group
inside of the aromatic ring of the phenylethyl part, whereas verapamil
contains two methoxy groups in meta- and para-
positions. D888 blocks the channel with higher affinity than verapamil
(about 300-fold) (Johnson et al., 1996), although verapamil is the only
drug in this class currently in clinical use. The levorotatory
()-enantiomers of PAAs are more potent than the dextrorotatory
(+)-enantiomers (Ferry et al., 1984). Here, the ()-isomers of
verapamil and its derivatives are considered, with clockwise
arrangement of aryl, isopropyl, and C
N groups around the chiral
center upward and the rest of the structure downward (Carey, 1996).
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Fig. 10.
Verapamil conformations. Verapamil is shown in
space-filling images in the extended (A), folded (B), and half-folded
(C) conformations.
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Verapamil can adopt three structural forms (or conformational shapes):
extended, folded, and half-folded. The energetically optimal
conformations of each shape are shown in Fig. 10. The folded conformation is stabilized by nonbonded interactions of dimethoxyaryl rings on the opposite ends of verapamil, and this should be the most
stable conformation of the isolated drug. The conformation of the drug
during its interaction with the channel is uncertain, making it less
useful in developing a model of the binding site. However, because PAAs
both block the pore and inhibit binding of DHP, we propose that during
its formation of a complex with the channel, the PAA drug adopts the
half-folded shape, allowing it to occlude the interface III/IV crevice
and simultaneously occupy the central pore.
The proposed docking of D888 in its half-folded conformation is shown
in Fig. 11. D888 predominately
interacts with transmembrane segment IVS6 (Hockerman et al., 1997b),
and makes immediate van der Waals contacts with the side chains of
three amino acid residues, Tyr-1463, Ala-1467, and Ile-1470, which have
been shown to be the most critical residues for PAA binding (Hockerman
et al., 1995). The aromatic ring of the phenylethylamine part of this derivative is located inside the interface III/IV crevice, where we
have also proposed that the DHP ring binds, thereby participating in
aromatic-aromatic interaction with Tyr-1463 (Fig. 11) with an energy of
nonbonded interaction of about 5 kcal/mol. This permits the isopropyl
substituent of the alkyl chain of D888 to be located between the side
chains of Ala-1467 and Ile-1470, whereas the methyl groups of both
methoxy groups, substituting for the second phenyl ring on the opposite
side, can reach and surround Ile-1470, and this phenyl ring is located
inside the pore. These contacts between D888 and IVS6 generate the
hydrophobic component of the potential energy of interaction between
the molecules. In this arrangement, the presence of the second
para-methoxy group in the aromatic ring of phenylethylamine
introduces interference with the side chains of residues in the back
wall of the interface crevice (Met-1464), which might lead to the
reduced binding affinity of verapamil relative to D888.
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Fig. 11.
Binding of D888. Binding of desmethoxyverapamil
(D888) in the half-folded conformation inside of the pore and the
interface crevice between domains III and IV. The aromatic rings on the
ends of D888 interact with the side chains of Tyr-1463 and Ile-1470,
correspondingly. The central amine interacts directly with the
carboxylate of the side chain of Glu-1118 of the selectivity filter,
thereby blocking permeation.
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The recent experimental finding that a mutation in the IIIS5 -helix
(T1039Y) has improved binding of devapamil (Huber et al., 2002)
supports the requirement that one of the aromatic rings of PAA in the
bound state occupies the III/IV interface crevice. Such modulation of
binding is possible if PAAs also occlude the space outside of the
central pore that is close to the back wall of our proposed DHP binding cavity.
In contrast to the DHP molecules, the PAA molecules are predominantly
positively charged at physiological pH because of the tertiary amine,
and therefore they could interact electrostatically with the only
negative charges in the central pore - the selectivity filter
glutamates. In the half-folded conformation that we have chosen for
docking (Fig. 11), the central amine is on the corner between two
perpendicular fragments of the PAA molecule. With the aromatic ring of
phenylethylamine inside the DHP binding pocket, this amine is directed
very close to the carboxylate group of the side chain of Glu-1118
(domain III) and also to that of Glu-1419 (domain IV) (Fig. 11), with
formation of corresponding ion pairs. Experimentally, substitution of
Glu-1118 and Glu-1419 by glutamines resulted in a reduction in D888
affinity by 20- and 15-fold, respectively (Hockerman et al., 1997a). In
terms of free energies, such changes correspond to about 1.5
kcal/mol. The same mutations produce only a 4-fold change in the
affinity of DHP (Peterson and Catterall, 1995). In the proposed
orientation of D888, its amine could also interact with the side chain
of Tyr-1463 (IVS6) and possibly with its hydroxyl (Fig. 11). According
to mutational data, the mutant Y1463F substantially disrupted block by
D888 (Johnson et al., 1996), which Hockerman et al. (1997a) suggested
was the result of a hydrogen bond between Tyr-1463 and D888. If the
amine is very close to Tyr-1463, then its methyl substituent is
directed to the side chain of Tyr-1152 (IIIS6), so Tyr-1152 would not
be important for PAA binding. Correspondingly, its replacement with Ala
did not decrease the affinity for D888 (Table 2). Therefore, in this
proposed model, PAA binding involves simultaneous hydrophobic and ionic
interactions with the selectivity filter, the interdomain III/IV
crevice, and the pore of the L-type Ca2+ channel.
General Model Features.
Spacial organization of the S5 and S6
transmembrane -helices of the Ca2+ channel on
the basis of the M1 and M2 segments of KcsA is plausible and has been
used by several investigators (Huber et al., 2000; Lipkind and Fozzard,
2001; Zhorov et al., 2001). However, careful alignment of the
-helices is critical if the residues facing the pore and the
adjacent S6 segments are to include the residues experimentally shown
to be required for drug interaction. The KcsA P loop is inappropriate
for the Ca2+ channel because of the need for
direct involvement of the side chains of the glutamate residues of the
selectivity filter in Ca2+ binding. In addition,
the KcsA P loops would cover most of the critical S6 residues involved
in the drug interaction. The exact location of the selectivity filter
is critical, because it represents an upper spacial limit for the drug
binding site and because drug affinities are influenced by ions in the
selectivity filter.
The KcsA channel coordinates probably correspond to a closed
conformation of the channel (Jiang et al., 2002b), so it is likely that
our modeled Ca2+ channel pore is also closed.
Little is known about the conformational changes that occur during
voltage-dependent L-type Ca2+ channel activation
and inactivation gating. Mutational studies suggest that the S4
segments from only domains I and III are involved in activation (Garcia
et al., 1997), and multiple regions seem to play a role in some type of
inactivation (Hering et al., 2000). A K+ channel
with a homologous two-transmembrane segment subunit structure (MthK),
which is open in the presence of low concentrations of Ca2+, has now been structurally determined by
X-ray crystallography and it is almost identical to that of KcsA (Jiang
et al., 2002a). However, the conformation of its M2 helices (analogous
to the S6 helices of the Shaker channel) is different from that of the KcsA channel, corresponding to an open state. In the MthK structure, the lower half of the M2 helix is bent outward, as the result of a
flexible hinge formed by a highly conserved glycine residue (Gly-83 in
MthK or Gly-99 in KcsA), thereby avoiding the helical crossover seen in
the KcsA channel and resulting in opening of the pore vestibule to a
diameter of 12 Å. At first glance, this seems to offer us the
opportunity to model the L-type Ca2+ channel in
its activated-open conformation, allowing us to compare changes of drug
interaction with gating. However, there is no structural equivalent to
the MthK hinge in the domains IIIS6 and IVS6 -helices because these
Ca2+ channel domains contain neither this glycine
residue nor helix-breaking prolines; instead, conformationally
restricted isoleucine or cysteine residues are present in the
corresponding positions (Table 3). Consequently,. The S6 segments of
domains I and II do contain the important glycine residue, which is
conserved in all isoforms of the Ca2+ channel
(see Stea et al., 1994, and our alignment for IS6 and IIS6 in Lipkind
and Fozzard, 2001). It is likely that gating of the
Ca2+ channel is different, with hinged motion of
domains I and II S6 -helices, whereas those of domains III and IV
remain predominantly straight. This supports our use of the KcsA
structure in modeling the core structure of domains III and IV for the
drug high-affinity state.
Although it is tempting to suggest that the modeled conformation with
crossover of the domains III and IV helices is close to the structure
of the normal inactivated state, drug interaction could induce a
different closed state. Berjukow et al. (2000) showed that alanine
substitution for the residues corresponding to Ile-1470 and Ile-1471 at
the crossover slowed the onset of inactivation. In addition,
substitution of some of the hydrophobic residues closer to the C-end of
IIIS6 can dramatically decrease the rate of inactivation (IF1612/1613AA
in IIIS6 of Cav2.1; Hering et al., 2000; Sokolov
et al., 2000). Both observations are consistent with our proposal that
DHP antagonists could interact with a closed state that resembles the
inactivated state and stabilize it by interaction at the level of the
IIIS6 and IVS6 helical crossover. If, as predicted by the model, DHP
antagonists bind to Ile-1163 and Ile-1471 with a total of 5 kcal/mol,
and this interaction occurs only in the inactivated state, then this
interaction could account for the difference in affinity between the
hyperpolarized, resting state and a depolarized state. We hypothesize
that the different affinities of the gating states can be mediated by
relatively small conformational changes inside the IIIS6/IVS6 binding
site, sufficient to disrupt short-range hydrophobic interactions.
Because of the similarity of DHP antagonist and agonist binding
interaction, we endorse the strong argument that DHP must not block the
pore (Hockerman et al., 1997b), whereas the PAA site must overlap and
simultaneously block the pore. This requires that DHP influence the
current by altering gating, which is more easily satisfied if the drug
binds close to the gating apparatus. We have suggested that this gating
effect might be mediated via interaction with the side chains of
hydrophobic residues at the S6 helix crossing point, because this is
the location in the binding site at which the critical difference
between antagonist and agonist DHP binding is found and at which
mutations produce changes in inactivation behavior of the L-type
Ca2+ channel, as well as in the similar
Na+ channel (McPhee et al., 1995; Sunami et al.,
2000). The interactions suggested by this model are amenable to test by
mutant cycle analysis between mutated channel residues and analogs of
the drugs, similar to studies of the binding interactions for
guanidinium toxins and µ-conotoxin in the vestibule of
Na+ channels (Chang et al., 1998; Dudley et al.,
2000; Penzotti et al., 2001).
| |
Conclusions |
To test the domain interface hypothesis for the formation of a
binding site for DHP and PAA drugs, we aligned the sequences of the
domain III S5 and S6 and domain IV S6 of the L-type
Ca2+ channel, using the spacial orientation of
the backbones of M1 and M2 -helices of the KcsA channel derived from
its X-ray crystal structure, maximizing the coincidence of residues
known to affect the drug biological activity with the pore-facing
residues of KcsA. The resulting structure formed a crevice between
domain III S6 and domain IV S6, with its top formed by the domain III P
loop and its back formed partly by domain III S5. DHPs were considered
in their preferred cis- conformation of the port side ester
group. For the optimal arrangement of a derivative of nifedipine, the
4-aryl ring interacted with the side chains of Tyr-1152 and Tyr-1463,
the DHP ring itself was located para
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Abstract
Introduction
