Structural model for phenylalkylamine binding to L-type calcium channels

Abstract

Phenylalkylamines (PAAs), a major class of L-type calcium channel (LTCC) blockers, have two aromatic rings connected by a flexible chain with a nitrile substituent. Structural aspects of ligand-channel interactions remain unclear. We have built a KvAP-based model of LTCC and used Monte Carlo energy minimizations to dock devapamil, verapamil, gallopamil, and other PAAs. The PAA-LTCC models have the following common features: (i) the meta-methoxy group in ring A, which is proximal to the nitrile group, accepts an H-bond from a PAA-sensing Tyr_IIIS6; (ii) the meta-methoxy group in ring B accepts an H-bond from a PAA-sensing Tyr_IVS6; (iii) the ammonium group is stabilized at the focus of P-helices; and (iv) the nitrile group binds to a Ca(2+) ion coordinated by the selectivity filter glutamates in repeats III and IV. The latter feature can explain Ca(2+) potentiation of PAA action and the presence of an electronegative atom at a similar position of potent PAA analogs. Tyr substitution of a Thr in IIIS5 is known to enhance action of devapamil and verapamil. Our models predict that the para-methoxy group in ring A of devapamil and verapamil accepts an H-bond from this engineered Tyr. The model explains structure-activity relationships of PAAs, effects of LTCC mutations on PAA potency, data on PAA access to LTCC, and Ca(2+) potentiation of PAA action. Common and class-specific aspects of action of PAAs, dihydropyridines, and benzothiazepines are discussed in view of the repeat interface concept.

FIGURE 1.

Structural formulae of PAAs.

FIGURE 2.

PAA-sensing residues and experimental constraints. S5s, P-helices, and S6s of the KvAP-based model of LTCC are shown as thin helices, ribbons, and thick helices, respectively. Repeats I and II are cyan, repeat III is green, and repeat IV is pink. Ca²⁺ ions are shown as yellow spheres. A and B, cytoplasmic and side views with PAA-sensing residues shown by surfaces. Repeat I in B is removed for clarity. C, intracellular view of the PAA-binding region. Ligand-sensing residues are shown as sticks and are labeled according to Table 1. Carbon atoms are colored as the backbones of corresponding repeats. D, proposed specific devapamil-LTCC interactions, which were used as constraints in modeling.

FIGURE 3.

Model of devapamil binding in LTCC. Rendering and coloring schemes are the same as described in the legend to Fig. 2. Devapamil is shown by sticks with dark-gray carbons. A and B, intracellular and side overviews. C and D, enlarged intracellular and side views. E, scheme of interactions between devapamil and PAA-sensing residues of LTCC in our model. Note the location of the ammonium group at the focus of P-helices and the interaction between the nitrile nitrogen and a Ca²⁺ ion in the selectivity filter.

FIGURE 4.

Devapamil-binding LTCC models built with different templates. A, cytoplasmic view on superimposed templates. The templates are arranged to minimize deviations of α-carbons at positions 3o14, 3i10, and 4i11, which correspond to the devapamil H-bonding tyrosines in our model. C^α–C^β bonds in these positions are shown as sticks colored as follows: KcsA, red; K_v1.2, orange; KvAP, blue; MthK, purple. The backbones of KvAP are shown as described in the legend to Fig. 2; other backbones are light gray. Note the relatively small variations in the C^α–C^β bonds among the four templates. B and C, intracellular and side views on the superposition of devapamil-binding models in the four LTCC homology models based on the four x-ray templates of K⁺ channels. The tyrosines are shown as thin sticks colored as the C^α–C^β bonds in A. Devapamil is shown by thick sticks with dark-gray carbon atoms. Calcium ions are shown as yellow spheres. The side chains of the engineered Y^3o14 are hidden in the side view (C) for clarity. Note that the variations in the templates cause only small variations in devapamil binding poses without breaking any specific contact. D, side view of the KcsA-based model of the closed LTCC. Repeats are shown as surfaces with repeat I removed for clarity. Devapamil is shown by sticks and a transparent surface. Note that the folded devapamil molecule matches the size of the central cavity.

FIGURE 5.

Comparison of S6 alignments. Shown are intracellular views of the KvAP template, with orange sticks indicating C^α–C^β bonds at positions that correspond to the H-bonding tyrosines of LTCC according to the different alignments of S6s. For IIIS5, the alignment proposed in Ref. was used. Orange lines define a triangle with the C^α atoms at the vertices. The triangle indicates the III/IV interface and defines a plane. A vector extending the C^α–C^β bond is colored blue or red if its projection on the plane directs into or out of the triangle, respectively. A–D correspond to the alignments proposed in Refs. , , , and , respectively. Only the alignment used in this work (D) results in orientation of all three vectors into the III/IV repeat interface and is therefore consistent with the proposed scheme of interactions.

FIGURE 6.

Different PAAs in the LTCC model. A, verapamil binding. The p-methoxy group of ring B occurs in the hydrophobic environment of A⁴ⁱ¹⁵ and I⁴ⁱ¹⁸ (shown as surfaces). B, gallopamil binding. The second m-methoxy group of ring A occurs in the hydrophobic environment of M³ⁱ¹⁸ and M³ⁱ¹⁹ (shown as surfaces). C, binding of (R)-devapamil. The isopropyl group (indicated by the arrow) faces the aqueous inner pore and makes no specific contacts. D, tiapamil binding. The bulky six-membered ring is readily accommodated in the pore. The orange arrows indicate the two oxygens that interact with the Ca²⁺ ion analogously to the nitrile group in classical PAAs. E, compound 2b from Ref. containing a cyclohexyl ring (indicated by the arrow) that restricts flexibility of the alkylamine chain. Despite the restricted flexibility, the compound readily adopts a U-shaped (54) conformation and preserves virtually all specific PAA-LTCC contacts.

FIGURE 7.

Binding of DHP (orange) and PAA (blue) in the III/IV repeat interface of LTCC. A, cytoplasmic view. Although DHPs and PAAs have overlapping binding sites (1), the binding modes independently predicted in our recent (20) and current studies show no significant overlap between the ligand molecules. B, side view with repeats II and IV removed for clarity. The PAA ligand reaches the binding site in the inner pore through the open activation gate. Only a small part of the ligand occupies the repeat interface. The DHP ligand reaches the binding site from the extracellular medium along the pore helix of repeat III. Only the hydrophobic moiety, which determines the antagonistic action of DHPs, extends to the pore. The engineered Y^3o14 (green space-filled) would sterically prevent the binding of the DHP ligand but provides an additional H-bonding contact for the PAA ligand in our model.