Introduction

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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.

	Materials and Methods

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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.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions References

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 Ca_v1.2 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 S6Tyr-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 K_d 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).

View this table: [in this window] [in a new window]   TABLE 2 Effects of alanine mutations on domains III and IV S6 segments of the L-type Ca2+ channel on binding of ()-D888

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 Ca_v1.2 residues (Table 3). Table 3 shows two possible alignments of the Ca_v1.2 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.

View larger version (78K): [in this window] [in a new window]   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.

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 Ca_v1.2 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.

View larger version (47K): [in this window] [in a new window]   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.

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.

View larger version (55K): [in this window] [in a new window]   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.

View larger version (8K): [in this window] [in a new window]   Fig. 4.   Chemical structure of dihydropyridines with the port side fused thiophene rings. A, active derivative. B, nonactive derivative (Adachi et al., 1988).

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.

View larger version (51K): [in this window] [in a new window]   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.

View larger version (52K): [in this window] [in a new window]   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.

View larger version (50K): [in this window] [in a new window]   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.

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.

View larger version (84K): [in this window] [in a new window]   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.

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).

View larger version (49K): [in this window] [in a new window]   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.

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.

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 CN groups around the chiral center upward and the rest of the structure downward (Carey, 1996).

View larger version (67K): [in this window] [in a new window]   Fig. 10.   Verapamil conformations. Verapamil is shown in space-filling images in the extended (A), folded (B), and half-folded (C) conformations.

View larger version (48K): [in this window] [in a new window]   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.

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.

	Conclusions

Top Abstract Introduction Materials and Methods Results and Discussion Conclusions References

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 parallel to the axis of the pore in the crevice depth, and the nonpolar isopropyl substituent of the ester group on the port side participated in strong interactions with the side chains of Ile-1156, Ile-1163, and Ile-1471 on the bottom. The model allowed an explanation of the different inhibitory activity of DHPs with fused thiophene rings on the port side with alkyl substitutions in the 3rd position (active) and in the 2nd position (inactive). DHP agonists adopted a similar arrangement inside the interface crevice, so that both agonists and antagonists were located outside the central pore, where they do not block by pore occlusion. However, the antagonists had nonpolar interactions with residues at the crossing of domains III and IV S6 helices at the bottom of the crevice that could prevent helical movements involved in activation by stabilizing the closed state; agonists in this conformation were not able to interact with the hydrophobic crossing residues, destabilizing the closed state. PAAs were docked in their half-folded conformation with the aromatic ring of the phenylethylamine part of D888 located in the interface III/IV crevice in proximity to the side chain of Tyr-1463 of IV S6, thereby blocking binding of DHP molecules. The second aromatic ring in the pore interacted with the side chain of Ile-1470. The central amine of D888 interacted directly by a salt bridge with the carboxylate side chain of Glu-1118 of the selectivity filter, blocking Ca²⁺ permeation.