Structural Model for Phenylalkylamine Binding to L-type Calcium Channels
2009; Elsevier BV; Volume: 284; Issue: 41 Linguagem: Inglês
10.1074/jbc.m109.027326
ISSN1083-351X
AutoresRicky C. Cheng, Denis B. Tikhonov, Boris S. Zhorov,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoPhenylalkylamines (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 Ca2+ ion coordinated by the selectivity filter glutamates in repeats III and IV. The latter feature can explain Ca2+ 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 Ca2+ 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. 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 Ca2+ ion coordinated by the selectivity filter glutamates in repeats III and IV. The latter feature can explain Ca2+ 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 Ca2+ 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. L-type calcium channels (LTCCs) 2The abbreviations used are: LTCCL-type calcium channelBTZbenzo(thi)azepineDHPdihydropyridinePAAphenylalkylamine. 2The abbreviations used are: LTCCL-type calcium channelBTZbenzo(thi)azepineDHPdihydropyridinePAAphenylalkylamine. are targets for different drugs. Benzo(thi)azepines (BTZs), dihydropyridines (DHPs), and phenylalkylamines (PAAs) constitute the three major classes of the LTCC ligands (for reviews, see Refs. 1Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar and 2Lacinova L. Gen. Physiol. Biophys. 2005; 24: 1-78PubMed Google Scholar). All of these ligands bind to overlapping binding sites in the pore-forming domain of the α1 subunit, but each class demonstrates unique characteristics of action. Depending on their chemical structure, DHPs act as agonists or antagonists (3Triggle D.J. Cell. Mol. Neurobiol. 2003; 23: 293-303Crossref PubMed Scopus (247) Google Scholar). All known PAAs and BTZs are antagonists, but they have different access pathways to their binding sites: external for BTZs (4Hering S. Savchenko A. Strübing C. Lakitsch M. Striessnig J. Mol. Pharmacol. 1993; 43: 820-826PubMed Google Scholar, 5Seydl K. Kimball D. Schindler H. Romanin C. Pflugers Arch. 1993; 424: 552-554Crossref PubMed Scopus (22) Google Scholar) and predominantly internal for PAAs (6Hescheler J. Pelzer D. Trube G. Trautwein W. Pflugers Arch. 1982; 393: 287-291Crossref PubMed Scopus (176) Google Scholar). Clinical use of verapamil in treatments of hypertension and arrhythmias (7Triggle D.J. J. Cardiovasc. Pharmacol. 1991; 18: S1-S6Crossref PubMed Scopus (78) Google Scholar) had stimulated intensive electrophysiological, mutational, and pharmacological studies involving PAAs. L-type calcium channel benzo(thi)azepine dihydropyridine phenylalkylamine. L-type calcium channel benzo(thi)azepine dihydropyridine phenylalkylamine. The pore-forming domain of LTCC includes the pore-lining inner helices S6, the outer helices S5, and the P-loops from all four repeats of the α1 subunit. According to mutational analyses, the PAA-binding site is located in the interface between repeats III and IV. In particular, residues in transmembrane helices IIIS5, IIIS6, and IVS6 and P-loops of repeats III and IV contribute to binding of PAAs (8Dilmac N. Hilliard N. Hockerman G.H. Mol. Pharmacol. 2004; 66: 1236-1247Crossref PubMed Scopus (28) Google Scholar, 9Döring F. Degtiar V.E. Grabner M. Striessnig J. Hering S. Glossman H. J. Biol. Chem. 1996; 271: 11745-11749Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 10Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 11Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12Huber I.G. Wappl-Kornherr E. Sinnegger-Brauns M.J. Hoda J.C. Walter-Bastl D. Striessnig J. J. Biol. Chem. 2004; 279: 55211-55217Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 13Schuster A. Lacinová L. Klugbauer N. Ito H. Birnbaumer L. Hofmann F. EMBO J. 1996; 15: 2365-2370Crossref PubMed Scopus (115) Google Scholar, 14Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar). Structure-activity relationships of PAAs were intensively studied (15Goll A. Glossmann H. Mannhold R. Naunyn-Schmiedeberg's Arch. Pharmacol. 1986; 334: 303-312Crossref PubMed Scopus (27) Google Scholar, 16Mannhold R. Holtje H.D. Koke V. Arch. Pharm. 1986; 319: 990-998Crossref PubMed Scopus (8) Google Scholar, 17Mannhold R. Steiner R. Haas W. Kaufmann R. Naunyn-Schmiedeberg's Arch. Pharmacol. 1978; 302: 217-226Crossref PubMed Scopus (51) Google Scholar). A common feature of potent PAAs is the presence of two methoxylated aromatic rings (named A and B). The rings are connected by a flexible alkylamine chain with a nitrile and an isopropyl group at the chiral tetrasubstituted carbon atom, which is proximal to ring A. Ring B is proximal to the amino group (see Fig. 1). Despite the fact that some specific contacts between functional groups of PAAs and PAA-sensing residues (residues that, when mutated, affect action of PAAs) have been proposed (10Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 14Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar), the flexibility of the ligands did not allow the characterization of the binding mode and the general pattern of ligand-channel interactions. In the absence of such knowledge, it is hardly possible to provide a molecular basis for structure-activity relationships. The problem is further complicated by the dependence of PAA action on the functional state of the channel, the ionic environment, the transmembrane voltage, and other factors. For example, it is generally believed that PAAs bind to the open/inactivated channels with higher affinities than to the closed state (for review, see Ref 1Hockerman G.H. Peterson B.Z. Johnson B.D. Catterall W.A. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 361-396Crossref PubMed Google Scholar). However, the molecular basis for this state dependence is unclear. Lipkind and Fozzard (18Lipkind G.M. Fozzard H.A. Mol. Pharmacol. 2003; 63: 499-511Crossref PubMed Scopus (63) Google Scholar) docked devapamil in a KcsA-based homology model of the L-type Ca2+ channel. They suggested an angular conformation of the drug, with ring B extended into the III/IV repeat interface and ring A in the central cavity. They also suggested that the protonated amino group of devapamil interacts directly with the selectivity filter glutamates. This model explains the effect of some mutations, particularly those in the P-loops and IVS6. However, other important aspects of PAA action such as the role of the nitrile group, the Ca2+ potentiation effect, and the effects of mutations in IIIS6 and IIIS5 remain unexplained. The gap between the amount of experimental data on PAA action and the level of understanding of the atomic level mechanisms necessitates further studies. In the absence of x-ray structures of Ca2+ channels, molecular modeling is the only available approach to address the structural aspects of PAA-LTCC interactions. Recently, we proposed molecular models for the action of other classes of L-type channel ligands. In the BTZ-LTCC models (19Tikhonov D.B. Zhorov B.S. J. Biol. Chem. 2008; 283: 17594-17604Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the main body of the ligands binds in the repeat interface, whereas the amino group protrudes into the inner pore, where it is stabilized by nucleophilic C-terminal ends of the pore helices. In the DHP-LTCC models (20Tikhonov D.B. Zhorov B.S. J. Biol. Chem. 2009; 284: 19006-19017Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), the ligands also bind in the interface between repeats III and IV, whereas the moieties that differ between agonist and antagonists extend to the pore. Both models suggest direct interactions between the ligands and a Ca2+ ion bound to the selectivity filter glutamates in repeats III and IV. In this work, we elaborate molecular models for PAA·LTCC complexes that agree with a large body of experimental data. We further discuss common and different aspects of action of different ligands on LTCC and propose that certain aspects of the ligand action may be relevant to other P-loop channels. Models were constructed using ZMM software. Energy was expressed as a sum of van der Waals, electrostatic, and torsional components as well as energy of deformation of bond angles of ligands. Bond angles of the channel were kept rigid. AMBER force field was used (21Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta S. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4881) Google Scholar, 22Weiner S.J. Kollman P.A. Nguyen D.T. Case D.A. J. Comput. Chem. 1986; 7: 230-252Crossref PubMed Scopus (3596) Google Scholar). To take into account that atomic charges may be screened by water molecules, electrostatic interactions were calculated with solvent exposure and distance-dependent dielectric function, ϵ = d(8 − 6s), where d is the distance between interacting atoms and s is a screening factor calculated using a modified algorithm of Lazaridis and Karplus (23Lazaridis T. Karplus M. Proteins. 1999; 35: 133-152Crossref PubMed Scopus (1116) Google Scholar). This screening factor varies from 0 for a pair of water-exposed atoms to 1 for a pair of protein-buried atoms. We have built four models of Cav1.2 that are based on the x-ray structures of K+ channels in the open (24Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1205) Google Scholar, 25Jiang Y. Lee A. Chen J. Ruta V. Cadene M. Chait B.T. MacKinnon R. Nature. 2003; 423: 33-41Crossref PubMed Scopus (1636) Google Scholar, 26Long S.B. Campbell E.B. Mackinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1844) Google Scholar) and closed (27Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5736) Google Scholar) states. The sequence alignment of Cav1.2 and K+ channels and the residue labeling scheme are shown in Table 1. The channel models include S5s, P-loops, and S6s from the four repeats. The P-helices and ascending limbs (positions p47–p57) were modeled using our P-loop domain model of Nav1.4 (28Tikhonov D.B. Zhorov B.S. Biophys. J. 2005; 88: 184-197Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In this model (as well as in all available models of Na+ and Ca2+ channels), the side chains of the selectivity filter residues at position p50 face the pore lumen and directly interact with permeant ions. When viewed from the extracellular side, repeats I–IV were arranged clockwise around the pore axis (29Dudley Jr., S.C. Chang N. Hall J. Lipkind G. Fozzard H.A. French R.J. J. Gen. Physiol. 2000; 116: 679-690Crossref PubMed Scopus (89) Google Scholar). Extracellular linkers, which are far from the PAA-binding site, were not modeled. Ionizable residues except the selectivity filter glutamates (positions p50) were modeled in their neutral forms. Two Ca2+ ions were loaded onto the selectivity filter to counterbalance the negative charges of the selectivity filter glutamates.TABLE 1Sequence alignment used for homology modeling of Cav1.2a A universal scheme was used to label residues according to their positions in the sequence alignment (45Zhorov B.S. Tikhonov D.B. J. Neurochem. 2004; 88: 782-799Crossref PubMed Scopus (103) Google Scholar).b–e Sequences are from the Protein Data Bank (codes 1BL8, 1LNQ, 1ORQ, and 2A79, respectively).f The sequence is from UniProt (accession number P15381). a A universal scheme was used to label residues according to their positions in the sequence alignment (45Zhorov B.S. Tikhonov D.B. J. Neurochem. 2004; 88: 782-799Crossref PubMed Scopus (103) Google Scholar). b–e Sequences are from the Protein Data Bank (codes 1BL8, 1LNQ, 1ORQ, and 2A79, respectively). f The sequence is from UniProt (accession number P15381). Initial conformations of side chains were assigned using the SCWRL3 program (30Canutescu A.A. Shelenkov A.A. Dunbrack Jr., R.L. Protein Sci. 2003; 12: 2001-2014Crossref PubMed Scopus (873) Google Scholar). Models were optimized using the Monte Carlo minimization protocol (31Li Z. Scheraga H.A. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 6611-6615Crossref PubMed Scopus (1276) Google Scholar). Monte Carlo minimization trajectories were terminated when 10,000 consecutive energy minimizations did not improve the apparent global minimum. During energy optimizations, structural similarity between the model and the template was maintained by "pin" constraints. A pin is a flat-bottom parabolic energy penalty function that allows for penalty-free deviations of α-carbons up to 1 Å from their respective positions in the template and imposes a penalty with the force constant of 10 kcal mol−1 Å−2 for larger deviations. Atomic charges at ligands were calculated using the AM1 method in MOPAC (32Dewar M.J. Zoebisch E.G. Healy E.F. Stewart J.J. J. Am. Chem. Soc. 1985; 107: 3902-3909Crossref Scopus (15060) Google Scholar). Models were visualized using PyMOL (33DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, Palo Alto, CA2002Google Scholar). Hands-free docking of highly flexible PAAs (Fig. 1) is not promising because of the limited precision of homology models of LTCC based on x-ray structures of K+ channels. Therefore, we have used available data on ligand-channel interactions to decrease the number of degrees of freedom of the system and thus to guide our calculations to solutions consistent with experimental data. PAA-sensing residues available from the published mutational data are shown in Table 1, and their positions in the KvAP-based homology model are shown in Fig. 2. Straightforward interpretation of these data is possible only for the IVS6 mutants. In the triple mutant Y4i11A/A4i15S/I4i18A of Cav1.2, the IC50 for devapamil block increases by 100-fold compared with that of the wild type. The single mutation Y4i11F of Cav1.2 increases the IC50 for devapamil block by ∼6-fold. However, channel block by verapamil and gallopamil are unchanged between the wild type and the triple mutant (11Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar). Furthermore, a devapamil derivative with a photoreactive moiety at the meta-position of ring B labels a fragment from IVS6 (34Striessnig J. Glossmann H. Catterall W.A. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 9108-9112Crossref PubMed Scopus (139) Google Scholar). Based on these data and on the fact that devapamil differs from verapamil only by a methoxy group in ring B, the side chain of Y4i11 is proposed to form an H-bond with the m-methoxy group on ring B of devapamil (10Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). PAAs also interact with IIIS6 residues. In particular, mutation Y3i10F of Cav1.2 results in an 18-fold increase in the IC50 for the resting block by devapamil (10Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). This suggests an H-bond between devapamil and the side chain hydroxyl of Y3i10. Because ring B of devapamil has only one methoxy group, which forms an H-bond with Y4i11, we propose that the side chain of Y3i10 forms an H-bond with a methoxy group in ring A of devapamil. Given the rather close disposition of the tyrosine residues in IIIS6 and IVS6 (Fig. 2) and the large length of the ligand in the extended conformation, only a folded devapamil molecule can form these two H-bonds simultaneously. It should be noted that alanine substitution of Y3i10 enhances the potency of devapamil, but this mutation strongly increases steady-state inactivation of the channel (10Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Taking into account the state-dependent action of devapamil (35Lee K.S. Tsien R.W. Nature. 1983; 302: 790-794Crossref PubMed Scopus (674) Google Scholar), the effect of the Y3i10A mutation on devapamil binding is likely indirect. The third constraint can be obtained from the study showing that the T3o14Y mutation enhances the potency of devapamil and verapamil but not that of gallopamil (12Huber I.G. Wappl-Kornherr E. Sinnegger-Brauns M.J. Hoda J.C. Walter-Bastl D. Striessnig J. J. Biol. Chem. 2004; 279: 55211-55217Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). In our model, T3o14 is located in the interface between repeats III and IV. The engineered Y3o14 can approach Y3i10 (Fig. 2). Devapamil has two methoxy groups in ring A and only one methoxy group in ring B. On the basis of these data, we suggest that the two methoxy groups in ring A form H-bonds with both Y3i10 and Y3o14. To form H-bonds with the above-mentioned tyrosines (Y4i11, Y3i10, and the engineered Y3o14), the aromatic rings of devapamil must reach into the III/IV repeat interface. However, the interface does not have enough room to accommodate the entire devapamil molecule. Thus, if both aromatic rings of devapamil bind in the III/IV repeat interface, the central part of the molecule, which contains the amino and nitrile groups, must reside in the inner pore. Indeed, some of the PAA-sensing residues in the P-loops, IIIS6, and IVS6 face the inner pore rather than the repeat interface. The model in which the amino group of devapamil is located in the inner pore and interacts with the selectivity filter glutamates directly (18Lipkind G.M. Fozzard H.A. Mol. Pharmacol. 2003; 63: 499-511Crossref PubMed Scopus (63) Google Scholar) does not explain the Ca2+ potentiation of PAA action and the importance of the nitrile group in PAAs. In the ion-conducting state of the channel, these selectivity filter glutamates are involved in the permeation process and bind the permeating ion directly. If devapamil binds to these glutamates, it must compete with permeating ions. However, PAAs block Ba2+ currents less effectively than they block Ca2+ currents; this effect is known as Ca2+ potentiation (8Dilmac N. Hilliard N. Hockerman G.H. Mol. Pharmacol. 2004; 66: 1236-1247Crossref PubMed Scopus (28) Google Scholar). Ca2+ ions have a higher affinity for the selectivity filter glutamates than Ba2+ ions (36Almers W. McCleskey E.W. J. Physiol. 1984; 353: 585-608Crossref PubMed Scopus (473) Google Scholar). Mutations in the selectivity filter region disrupt the Ca2+ potentiation effect (8Dilmac N. Hilliard N. Hockerman G.H. Mol. Pharmacol. 2004; 66: 1236-1247Crossref PubMed Scopus (28) Google Scholar). Thus, the experimental data allow us to suggest that high affinity binding of PAAs requires the presence of a permeating ion bound to the selectivity filter. In other words, the Ca2+ ion in the selectivity filter would enhance the channel-blocking activity of PAAs. Such enhancement seems impossible in the model in which the amino group of PAAs binds directly to the selectivity filter glutamates. How can the binding of a positively charged metal ion to the selectivity filter enhance the binding of a positively charged PAA ligand? There are two possibilities: by an allosteric effect or by a direct PAA-Ca2+ interaction. We cannot rule out the first possibility, but a direct interaction seems more plausible because the ligand-binding site is close to the selectivity filter. Indeed, direct interactions between ions in the selectivity filter and a ligand in the inner pore were demonstrated for K+ channels by a combination of x-ray crystallography and high level free energy calculations (37Faraldo-Gómez J.D. Kutluay E. Jogini V. Zhao Y. Heginbotham L. Roux B. J. Mol. Biol. 2007; 365: 649-662Crossref PubMed Scopus (53) Google Scholar). If the Ca2+ potentiation effect is due to direct interaction with the ligand, the latter should contain functional group(s) capable of coordinating the Ca2+ ion. Because internally applied PAAs with tertiary and quaternary amino groups exhibit similar channel-blocking potencies (6Hescheler J. Pelzer D. Trube G. Trautwein W. Pflugers Arch. 1982; 393: 287-291Crossref PubMed Scopus (176) Google Scholar, 38Berjukov S. Aczel S. Beyer B. Kimball S.D. Dichtl M. Hering S. Striessnig J. Br. J. Pharmacol. 1996; 119: 1197-1202Crossref PubMed Scopus (13) Google Scholar), the tertiary amino group is likely protonated and hence has no lone electron pair. Methoxy groups have lone pairs, but these groups are involved in specific interactions with PAA-sensing residues of the channel (see above). The only remaining candidate for a direct coordination with a Ca2+ ion is the nitrogen atom in the nitrile group. This group is critically important for PAA action (16Mannhold R. Holtje H.D. Koke V. Arch. Pharm. 1986; 319: 990-998Crossref PubMed Scopus (8) Google Scholar), but its role in ligand-channel interactions has not been explained so far. The geometry of the nitrile group with the lone pair extending from the ligand along the triple-bond direction seems ideal for reaching the Ca2+ ion bound to the selectivity filter glutamates. All these considerations suggest that the nitrile group of PAAs interacts directly with the ion at the selectivity filter. If the amino group of PAAs does not bind to the selectivity filter glutamates, where can it bind? In addition to the selectivity filter, the P-loop channels have another site that attracts cations. This site is located at the focus of P-helices. In K+ channels, the macrodipoles of P-helices create a binding site for permeating ions and positively charged tetraalkylammonium ligands (39Gouaux E. Mackinnon R. Science. 2005; 310: 1461-1465Crossref PubMed Scopus (705) Google Scholar, 40Zhou M. Morais-Cabral J.H. Mann S. MacKinnon R. Nature. 2001; 411: 657-661Crossref PubMed Scopus (493) Google Scholar). In summary, the above analysis of experimental data suggests the following scheme of devapamil binding to LTCC: (i) the m-methoxy group in ring B accepts an H-bond from Y4i11, (ii) the two methoxy groups in ring A accept H-bonds from Y3i10 and the engineered Y3o14, (iii) the nitrile nitrogen coordinates a Ca2+ ion bound to the selectivity filter glutamates from repeats III and IV, and (iv) the protonated amino group is located in the nucleophilic region at the focus of P-helices (Fig. 2). The proposed binding scheme for devapamil is based on the data from mutational, electrophysiological, and ligand binding experiments (8Dilmac N. Hilliard N. Hockerman G.H. Mol. Pharmacol. 2004; 66: 1236-1247Crossref PubMed Scopus (28) Google Scholar, 10Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 11Hockerman G.H. Johnson B.D. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 22119-22122Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12Huber I.G. Wappl-Kornherr E. Sinnegger-Brauns M.J. Hoda J.C. Walter-Bastl D. Striessnig J. J. Biol. Chem. 2004; 279: 55211-55217Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 14Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar, 16Mannhold R. Holtje H.D. Koke V. Arch. Pharm. 1986; 319: 990-998Crossref PubMed Scopus (8) Google Scholar). It remains to be explored if this scheme agrees with available structures of P-loop channels, conformational possibilities of PAAs, and experimental data on the interactions of PAAs with the channel residues. We modeled devapamil binding to the T3o14Y mutant of the KvAP-based model of Cav1.2. The mutant contains more "anchors" to dock devapamil than the wild-type channel. Proximity between methoxy groups of devapamil and hydroxy groups of Y3o14, Y3i10, and Y4i11 was established by distance constraints between specific pairs of atoms. Distance constraints were also used to bias coordination of a Ca2+ ion by the nitrile group of devapamil as well as by E3p50 and E4p50. In addition to the six distance constraints, pin constraints (see "Materials and Methods") were imposed to retain the structural similarity of the channel model to the x-ray template. The complex was optimized by the Monte Carlo minimization protocol. The goal of this optimization was to check if all proposed interactions are energetically possible. A ligand-binding pose was considered possible if after Monte Carlo minimization it met the following four criteria: (i) all distance constraints were satisfied; (ii) the complex remained stable upon a subsequent energy minimization with all constraints removed, i.e. the complex corresponded to an energy minimum; (iii) the ligand-receptor energy was negative; and (iv) no amino acid in the ligand-binding site provided repulsive (positive) contributions of van der Waals energy to ligand-receptor interactions. The proposed binding scheme contains an ambiguity: from the experimental data, it is unclear which of the two methoxy groups in ring A H-bonds with Y3i10 and with Y3o14. Both possibilities were explored. Computations have shown that just one binding scheme meets the above criteria. In this scheme, p- and m-methoxy groups accept H-bonds from Y3o14 and Y3i10, respectively. The obtained binding mode is shown in Fig. 3. In this complex, the ligand-channel interaction energy is −41 kcal/mol. No residue provides unfavorable (positive) energy contribution (Table 2). Despite the fact that the ligand bears a net positive charge of 1 elementary charge unit, the Ca2+ ion provides a stabilizing contribution of −2 kcal/mol. This is not surprising: the nitrile group is strongly attracted to the Ca2+ ion, whereas the ammonium nitrogen is 6.2 Å from the Ca2+ ion. The ammonium group of devapamil does not form a direct contact with any residue, but it is stabilized by electrostatic interactions with the nucleophilic C-terminal ends of the four P-loops. The positively charged ammonium group of the ligand at the pore center appears as the major determinant of the ion permeation block. Ring B interacts with Y4i11, A4i15, and I4i18, which were described previously as the PAA-sensing triad (10Hockerman G.H. Johnson B.D. Abbott M.R. Scheuer T. Catterall W.A. J. Biol. Chem. 1997; 272: 18759-18765Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 14Johnson B.D. Hockerman G.H. Scheuer T. Catterall W.A. Mol. Pharmacol. 1996; 50: 1388-1400PubMed Google Scholar). Ring A interacts with F3p49, I3i11, and T3p48. The isopropyl group approaches T2p48 and F3p49. The central part of devapamil is located in the inner pore.TABLE 2Interactions of devapamil with Cav1.2 residuesResidueaListed are residues that have at least one side chain heavy atom within 5 Å from a heavy atom of devapamil and residues whose mutations were demonstrated to have significant affect on PAA action (8, 10, 11, 43). The latter residues are shown in boldface.Closest to devapamil side chain heavy atomSide chain devapamil energyPDB nameDistancebThe distance is between the indic
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