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May 1, 2008 - of hERG channels can cause the drug-induced form of long QT ... this cardiotoxic side effect has been a common reason for the failure of...
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Chem. Res. Toxicol. 2008, 21, 1005–1010

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hERG Potassium Channels and the Structural Basis of Drug-Induced Arrhythmias John S. Mitcheson* Department of Cell Physiology and Pharmacology, UniVersity of Leicester, Medical Sciences Building, UniVersity Road, Leicester, LE1 9HN, United Kingdom ReceiVed January 25, 2008

hERG potassium channels have a critical role in the normal electrical activity of the heart. The block of hERG channels can cause the drug-induced form of long QT syndrome, a cardiac disorder that carries an increased risk of cardiac arrhythmias and sudden death. hERG channels are extraordinarily sensitive to block by large numbers of structurally diverse drugs. In previous years, the risk of compounds causing this cardiotoxic side effect has been a common reason for the failure of compounds in preclinical safety trials. Pharmaceutical companies have successfully utilized and developed higher throughput techniques for the early detection of compounds that block hERG, and this has helped reduce the number of compounds that fail in the late stages of development. Nevertheless, this screening-based approach is expensive, consumes chemistry resources, and bypasses the problem rather than shedding light on it. Crystal structures of potassium channels have facilitated studies into the structural basis for the gating and block of hERG channels. Most drugs bind within the inner cavity, and the individual amino acids that form the drug binding site have been identified by site-directed mutagenesis approaches. Gating processes have an important influence on the drug-binding site. Recent advances in our understanding of channel activation and inactivation are providing insight into why hERG channels are more susceptible to block than other K+ channels. Knowledge of the structure of the drug-binding site and precise nature of interactions with drug molecules should assist efforts to develop drugs without the propensity to cause cardiac arrhythmias. Contents Introduction HERG Channel Structure Drugs Bind within the Inner Cavity of hERG Structure of the Drug-Binding Site Why Is hERG so More Sensitive to Block Than hEAG? 6. Conclusions 1. 2. 3. 4. 5.

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1. Introduction The hERG (human ether-a-go-go-related gene) or Kv11.1 potassium (K+) channel is one of a number of ion channels that contribute to the electrical activity of the human heart. The important role of hERG channels in cardiac function became evident when inherited mutations were found to cause long QT syndrome (LQTS), a disorder that leads to a delay in cardiac action potential repolarization and predisposes patients to potentially fatal arrhythmias (1). Drug-induced LQTS is more common than the inherited form and is a side effect of both cardiac and noncardiac medications (2). The drug-induced disease can result from direct block of channel conduction (3), disruption of channel trafficking to the surface (4-6), or indirect mechanisms such as inhibition of cytochrome P450 and a reduction in drug metabolism by the liver (7). Although the incidences of life-threatening arrhythmias are rare, it became evident that medications for even the most benign conditions could cause sudden death (8). This led to the withdrawal of certain medications by drug regulatory agencies worldwide. It * To whom correspondence should be addressed. Tel: +44(0)116 229 7133. Fax: +44(0)116 252 5045. E-mail: [email protected].

has now become routine for compounds to be tested for effects on hERG channel function early in drug development. Great progress has been made in developing techniques for preclinical testing of compounds for hERG-associated cardiotoxicity, and this has improved the safety profile of new medications. However, strategies for removing hERG liability remain elusive, and this problem continues to have a negative impact on drug development. Compounds that block hERG are frequently not taken further through development. As a consequence of the extraordinary pharmacological promiscuity of hERG, large numbers of potentially useful compounds are essentially discarded. In another perspective in this issue, by Maurizio Taglialatella, cardiotoxicity associated with antihistamines has been used to illustrate the mechanisms and problems associated with QT prolongation and arrhythmogenesis. In this perspective, I attempt to address the question of why the block of hERG channels in particular causes drug-induced arrhythmia by reviewing the structural basis of high affinity block of hERG channels.

2. HERG Channel Structure X-ray crystal structures of bacterial K channels [KcsA (9), MthK (10), and KvAP (11)] and of a eukaryotic Kv1.2 channel (12) have provided tremendous insight into the structural basis of gating and K+ ion permeation. hERG is a member of the Kv family of voltage-gated potassium channels. A crystal structure for the hERG channel is not currently available, but the basic architecture is likely to be similar to Kv1.2. Kv channels are formed by the coassembly of four subunits, each of which has six transmembrane spanning R-helices (referred to as S1-S6). The first four helices (S1-S4) fold to form four separate voltage-

10.1021/tx800035b CCC: $40.75  2008 American Chemical Society Published on Web 05/01/2008

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Figure 1. Homology models of hERG displaying the inner cavity amino acids suggested to form the drug-binding site. Left panel, closed state model based upon KcsA; right panel, open state model based on KvAP. The binding site residues are as follows: Val625, purple; Ser624, brown; Thr623, black; Gly648, orange; Tyr652, dark blue; Phe656, red; and Val659, pale blue.

Figure 2. Sequence alignment of pore domains of potassium channels. The asterisk indicates putative glycine hinge. Red boxes highlight aromatic residues important for drug binding in hERG. Numbering relates to the hERG sequence.

sensing domains that function to detect membrane potential (13). These are arranged around a central potassium-selective pore domain formed by coassembly of S5 and S6 from each subunit (Figure 1). The structures of the voltage-sensing domains and the voltage-dependent conformational changes that they undergo during channel gating are the subject of intense investigation (reviewed in refs 14 and 15) but are largely beyond the scope of this perspective. The K+ channel pore (Figure 1) consists of a narrow selectivity filter on the extracellular side that has evolved for rapid flux of K+ ions. The selectivity filter is a highly conserved sequence of amino acids (Thr/Ser-Val-GlyTyr/Phe-Gly; see Figure 2) that coordinate dehydrated K+ ions, separated by a water molecule, as they pass through in single file (9). The selectivity filter is held in position by a network of interactions with the pore helices and residues that form the outer mouth of the pore. Below the selectivity filter, the pore widens to form a water-filled vestibule called the inner cavity. The inner cavity is lined by residues from the C-terminal ends of the pore helices and by residues from S6. In the closed state, the cavity is relatively small and the S6 helices bundle together at the intracellular end of the pore to form a narrow aperture that is sufficiently small to restrict the movement of K+ ions. However, in the open state, bending of S6 enables the C-terminal ends of the helices to move apart, dramatically increasing the diameter of the aperture to allow movement of ions (10-12). Thus, the C-terminal ends of S6 form an intracellular barriersthe

activation gatesthat regulates access of ions across the pore. It was thought that a conserved Gly (indicated by an asterisk in Figure 2) functions as a hinge for activation gating (16-18). However, more recently, studies on hERG and inward rectifier K+ channels indicate that the role of the conserved Gly is to restrict interactions with neighboring residues on the pore helices in this tightly packed and functionally important region (19, 20). The flexibility for bending occurs at this position, but Gly is not obligatory and other residues can fulfill this function. Another consequence of the opening of the activation gate in hERG is that the splaying apart of the cytoplasmic ends of S6 increases the size and alters the shape of the inner cavity, and this has important consequences for drug binding.

3. Drugs Bind within the Inner Cavity of hERG The activation gate acts to not only restrict movement of K+ ions across the pore but also access of drug molecules to their binding site within the inner cavity. Compounds cross the plasma membrane, act from the cytoplasmic side of the channel, and only gain access to their binding site when the channel has opened (open channel block) (21, 22). Many high and low affinity compounds only recover from block relatively slowly (e.g., see refs 23-26). The explanation is that compounds are trapped within the inner cavity by closure of the activation gate upon repolarization (24). Recovery from block can be facilitated

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at negative potentials in the D540K hERG mutant because of its unusual characteristic of opening with hyperpolarization (as well as with depolarization), permitting trapped drug molecules to exit from the cavity into the cytoplasm (23, 24, 26, 27). The drug-trapping phenomenon is revealing because it indicates that the inner cavity of hERG is sufficiently large to accommodate large and structurally diverse compounds without obstructing movement of the activation gate. This is in contrast to findings on other channels, where drug unbinding must precede closure of the activation gate unless the blocker is extremely small (28). A structural explanation for the large size of the hERG channel inner cavity is not entirely clear. Kv channels have a conserved Pro-X-Pro motif on S6 that is absent in hERG and may act as an alternative gating hinge (29, 30), preventing drug binding in the closed state. It may also be explained by more subtle differences in how inner cavity residues are orientated in different gating states. Perhaps in the closed state, inner cavity residues are not optimally positioned for high affinity binding. A crystal structure of a Kv channel with the Pro-X-Pro motif in the closed state is needed to resolve this issue. Not all drugs exhibit this slow recovery from block kinetics (26). Recently, we have studied some derivatives of E4031 that are more potent than the parent compound, bind within the inner cavity, but appear to rapidly recover from block even when channels are closed. At present, it is not clear how this is achieved, but these results and others (26) cast doubt on the drug-trapping hypothesis and suggest that alternative mechanisms of block may exist. Despite this, there is little doubt that the inner cavity of hERG channels can physically accommodate a wide array of structurally diverse compounds as compared to other K+ channels.

4. Structure of the Drug-Binding Site The sheer number and diversity of compounds that block hERG channels suggest it has an unusual binding site. Alascanning mutagenesis methods have been used to identify the hERG residues that interact with a variety of compounds (31). Individual residues within the pore of the channel were substituted to Ala, and the resulting mutants were tested for sensitivity to hERG blockers. Mutation of two aromatic residues (Tyr652 and Phe656) located on S6 was found to dramatically reduce the potency of a number of high and low affinity blockers from different chemical and therapeutic classes (23, 27, 31-37). Sequence alignments illustrate that these residues are not conserved in K+ channels. In fact, most K+ channels have aliphatic Ile or Val residues in these positions, and only other members of the ether-a-go-go family have aromatic residues in homologous positions. This discovery helps explain the astonishing pharmacological sensitivity of hERG channels. Aromatic residues are capable of hydrophobic and electrostatic interactions. An interesting study by Fernandez et al. (38) demonstrated that cisapride and terfenadine required an aromatic residue at position 652 for potent block, suggesting that the π-electrons of Tyr interact either with the charged amine through cation-π interactions or with an aromatic group by π-stacking interactions. In contrast, at position 656, there was a convincing correlation between blocking potency and indices of hydrophobicity for substituted residues (38). Phe656 appears to be the more important site of interaction for most hERG blockers. Only a few compounds have been identified that do not appear to bind at this position (39, 40). In contrast, in a recent study utilizing an automated planar electrode patch clamp system to generate a consistent data set, we were surprised to find that half of 24 LQT compounds that we tested were insensitive to the Y652A mutation (41). Initial analysis suggests that molecules

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with an extended conformation and the highest affinity for hERG have a high dependency for Tyr652 while compounds with a more compact conformation are less sensitive to the Tyr mutationsperhaps because they are able to reorientate themselves and find alternative energetically favorable binding modes. Two residues on the pore helices (Thr623 and Ser624) are also important for binding of many compounds (31, 33, 34). These residues may be particularly important for compounds with polar or electronegative parasubstituents on the phenyl ring. Cooperative interations are suggested by the observation that mutating either one individually produced modest (∼50-fold) increases of IC50, whereas mutating both produced far larger increases. Mutation of Thr623 and Ser624 reduced the potency of a nitro-phenyl derivative of ibutilide by more than 1000fold. Thr623 and Ser624 lie close to the selectivity filter and are highly conserved in K+ channels. Although they are not unique to hERG and may not explain the chemical diversity of hERG blockers, in my view, they are an important component of the binding site. Together with the aromatic residues, they create a highly favorable environment for drug binding in which compounds can form multiple interactions with residues in the channel. The reason that hERG is blocked by such a diversity of compounds is that the inner cavity is lined by eight aromatic and eight polar residues that can interact with molecules by various means. A potential caveat of the mutagenesis approach is that a residue substitution may decrease the potency of a compound through an allosteric effect on drug binding rather than by disruption of a direct interaction (31, 42). Mutagenesis studies have identified several residues that are unlikely to form part of the drug-binding site but, when mutated, reduce the potency of compounds (43). Val659 is located less than one helical turn away from Phe656. The V659A mutation reduces the potency of most compounds that have been studied. However, the mutation also profoundly alters gatingsshifting the voltage dependence of activation by -30 mV and profoundly slowing deactivation. Recent work in our laboratory indicates that this mutation reduces potency by increasing rates of recovery from block between pulses. Gly648 mutations reduce the potency of many high affinity blockers such as dofetilide, ibutilide, clofilium, nifekalant, vesnarinone, and MK-499 but not other compounds such as cisapride, terfenadine, bepridil, and propafenone (23, 31-34). The absence of an effect for some compounds, but a >50 fold reduction of potency for others, suggests a highly specific role for Gly648. It is located close to the selectivity filter, in a tightly packed and functionally important region of the channel (19). A larger residue in this position may alter the positioning of other inner cavity residues (44)sa similar mechanism has been suggested for Val625, which when mutated is likely to reposition Ser624 (34). A pocket may also be present in the wild-type channel that may be required for optimal binding of the G648A mutant sensitive compounds and is reduced in volume by substitution to larger residues (31). Understanding the precise mechanisms by which mutations alter potency may suggest strategies that could help reduce the binding affinity of a compound against hERG. Thus, it would be useful to screen a library of compounds against G648A and S624A to build up a more detailed knowledge of what features render compounds sensitive to these mutations that could be used to guide compound development. To date, there is little information of this type in the public domain.

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5. Why Is hERG so More Sensitive to Block Than hEAG? The pore of the ether-a-go-go (EAG) K+ channel has considerable sequence homology with hERG. Indeed, sequence alignments indicate that EAG channels contain all of the binding site residues discussed above (Figure 2). Despite this, the potencies of most compounds are greatly reduced in EAG as compared to hERG (43, 45-47). Clofilium is one of a few exceptions, with a similar affinity for both channels (46). Chen et al. postulated that differences in the rotation of S6 might be sufficient to change the orientation of the aromatic residues relative to the inner cavity in EAG as compared to hERG (48). In this model, the aromatic residues of EAG are not optimally positioned for interacting with blockers. Consistent with this idea, repositioning aromatic residues one position down (which in R-helices reorientates the residues by ∼100°) increased the sensitivity of EAG channels to cisapride. In our original closed and open state homology models of hERG, there tended to be a poor agreement between the models and the mutagenesis data (44). Two residues that are not involved in drug binding (Ser649 and Ser660) are located in suitable positions to interact with bound drugs, whereas Tyr652 and Phe656 were not. Rotation of S6, so that the aromatic residues faced into the pore, produced docking poses with higher binding scores and a greater conformity with the mutagenesis data (44). Each of these studies suggest that the orientation of inner cavity residues is different in hERG as compared to EAG and crystal structures of KcsA, MthK, and KvAP and raises the intriguing possibility of a rotation of S6 with gating. C-type inactivation is a physiologically important gating process that regulates the amplitude of the hERG current during the cardiac action potential by rapidly altering K+ ion flux through the selectivity filter (49-51). Its importance is graphically illustrated by a recently identified short QT syndrome in patients with the N588K hERG mutation, which reduces inactivation and is associated with increased risk of arrhythmia and sudden death (52, 53). Several studies have indicated a link between inactivation gating and high affinity block (43, 45, 54, 55). Whereas inactivation is absent in EAG, hERG channels exhibit a rapid form of this gating process. Relatively little is known about the conformational rearrangements that occur with inactivation in voltage-gated channels. The gating process involves instability of the open selectivity filter and molecular dynamics simulations by Phillip Stansfeld in collaboration with Prof Mike Sutcliffe (University of Manchester) point to a role for a hydrogen bond network behind the selectivity filter that can be disrupted by mutations at the mouth of the channel and on the pore helices. In KcsA, the number and strength of hydrogen bonds between Glu71 on the pore helix and Asp80 on the extracellular side of the selectivity filter have been proposed to determine the rate and extent of inactivation (56-58). In addition to local conformational changes, rearrangements in other domains of the channel are likely and there is growing evidence that rotation or other movements of S6 occur (59). An inactivation-associated reorientation of S6 residues to positions that are most suitable for drug binding could explain the link between inactivation and binding affinity and the relative insensitivity of EAG channels to hERG blockers. The binding of compounds to the inner cavity of hERG is highly state-dependent. As described earlier, the activation gate must open for drugs to gain access to the inner cavity, and closure of the activation gate seems to be sufficient to trap some drug molecules within the inner cavity. However, there are other drug molecules that exhibit rapid recovery from block when

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the channels are closed and even in the continued presence of drug (26). Available evidence indicates that the binding affinity of the channel is much lower in the closed than the open and inactivated state, but how some drugs are able to dissociate from the closed state remains an interesting and unanswered question. Indeed, this is reminiscent of experiments in the late 1970s studying recovery of sodium channels from block by local anesthetics, in which hydrophobic drugs seemed to be able to unbind from closed channels (60).

6. Conclusions It has been estimated that 2-3% of all drug prescriptions include medications with the potential to induce LQTS (61). Although hERG block is not the only mechanism that may cause QT prolongation, it is likely to be the most important, and most if not all drugs with a QT liability block hERG currents at a therapeutically relevant concentration. Understanding the structural basis of hERG channel block offers the best hope of developing strategies for reducing hERG binding affinity of novel compounds without sacrificing primary target efficacy. The location of the principal drug binding site has been located, and the most important residues that interact with compounds have been identified. Nevertheless, it is clear that compounds do not all bind in the same manner. As we learn more about the gating of hERG and as molecular modeling becomes more sophisticated, it is becoming clear that structure of the binding site is complex and dynamic. The state dependence of block clearly has an impact on the estimation of IC50 and highlights the value of preclinical screening that utilizes voltage clampbased methods to reproduce voltage-dependent gating processes during the cardiac action potential. The challenge for the future is to determine the structure of the inner cavity of hERG in the closed, open, and inactivated state, to accurately model the flexibility and dynamics of the important binding site residues, and to identify the different binding modes that compounds adopt within the pore. Integration of information from in vitro hERG assays and computational models of hERG and its ligands (3, 44, 62-65) is likely to hold the key to understanding and evading the pharmacological promiscuity of hERG. Acknowledgment. Work in the laboratory is currently supported by the British Heart Foundation and BBSRC, as well as by studentship support from Novartis Horsham Research Centre and Pfizer Global Research and Development. Thanks go to past and present members of my laboratory in Leicester who have contributed to studies described in this perspective, particularly Dr. Matthew Perry, Dr. Rachael Hardman, Dr. Sarah Nelson, Dr. Phillip Stansfeld, Sarah Dalibalta, and Michael Chang. Phillip Stansfeld also made the figures. Thanks are also owed to Prof. Michael Sutcliffe (University of Manchester) for cosupervision of in silico modeling projects and to Martin Gosling (Novartis Horsham Research Centre) and Joanne Leaney (Pfizer Global Research and Development, Sandwich, United Kingdom) for support with supervision and funding of studentships as well as for providing valuable input into the projects.

References (1) Curran, M. E., Splawski, I., Timothy, K. W., Vincent, G. M., Green, E. D., and Keating, M. T. (1995) A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80, 795– 803. (2) Roden, D. M., Lazzara, R., Rosen, M., Schwartz, P. J., Towbin, J., and Vincent, G. M. (1996) Multiple mechanisms in the long-QT

PerspectiVe

(3)

(4) (5)

(6)

(7) (8) (9)

(10) (11) (12) (13) (14) (15) (16) (17) (18) (19)

(20) (21) (22)

(23) (24) (25)

(26) (27)

(28)

syndrome. Current knowledge, gaps, and future directions. The SADS Foundation Task Force on LQTS. Circulation 94, 1996–2012. Recanatini, M., Poluzzi, E., Masetti, M., Cavalli, A., and De Ponti, F. (2005) QT prolongation through hERG K(+) channel blockade: Current knowledge and strategies for the early prediction during drug development. Med. Res. ReV. 25, 133–166. Eckhardt, L. L., Rajamani, S., and January, C. T. (2005) Protein trafficking abnormalities: A new mechanism in drug-induced long QT syndrome. Br. J. Pharmacol. 145, 3–4. Kuryshev, Y. A., Ficker, E., Wang, L., Hawryluk, P., Dennis, A. T., Wible, B. A., Brown, A. M., Kang, J., Chen, X. L., Sawamura, K., Reynolds, W., and Rampe, D. (2005) Pentamidine-induced long QT syndrome and block of hERG trafficking. J. Pharmacol. Exp. Ther. 312, 316–323. Wible, B. A., Hawryluk, P., Ficker, E., Kuryshev, Y. A., Kirsch, G., and Brown, A. M. (2005) HERG-Lite: A novel comprehensive highthroughput screen for drug-induced hERG risk. J. Pharmacol. Toxicol. Methods 52, 136–145. Dumaine, R., Roy, M. L., and Brown, A. M. (1998) Blockade of HERG and Kv1.5 by ketoconazole. J. Pharmacol. Exp. Ther. 286, 727–735. Viskin, S. (1999) Long QT syndromes and torsade de pointes. Lancet 354, 1625–1633. Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) The structure of the potassium channel: Molecular basis of K+ conduction and selectivity. Science 280, 69–77. Jiang, Y. X., Lee, A., Chen, J. Y., Cadene, M., Chait, B. T., and MacKinnon, R. (2002) The open pore conformation of potassium channels. Nature 417, 523–526. Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. T., and MacKinnon, R. (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41. Long, S. B., Campbell, E. B., and Mackinnon, R. (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903. Long, S. B., Campbell, E. B., and Mackinnon, R. (2005) Voltage sensor of Kv1.2: Structural basis of electromechanical coupling. Science 309, 903–908. Swartz, K. J. (2004) Towards a structural view of gating in potassium channels. Nat. ReV. Neurosci. 5, 905–916. Chanda, B., and Bezanilla, F. (2008) A common pathway for charge transport through voltage-sensing domains. Neuron 57, 345–351. Yifrach, O., and MacKinnon, R. (2002) Energetics of pore opening in a voltage-gated K(+) channel. Cell 111, 231–239. Ding, S., Ingleby, L., Ahern, C. A., and Horn, R. (2005) Investigating the putative glycine hinge in Shaker potassium channel. J. Gen. Physiol. 126, 213–226. Magidovich, E., and Yifrach, O. (2004) Conserved gating hinge in ligand- and voltage-dependent K+ channels. Biochemistry 43, 13242– 13247. Hardman, R. M., Stansfeld, P. J., Dalibalta, S., Sutcliffe, M. J., and Mitcheson, J. S. (2007) Activation gating of hERG potassium channels: S6 glycines are not required as gating hinges. J. Biol. Chem. 282, 31972–31981. Rosenhouse-Dantsker, A., and Logothetis, D. E. (2006) New roles for a key glycine and its neighboring residue in potassium channel gating. Biophys. J. 91, 2860–2873. Mitcheson, J. S., and Sanguinetti, M. C. (1999) Biophysical properties and molecular basis of cardiac rapid and slow delayed rectifier potassium channels. Cell. Physiol. Biochem. 9, 201–216. Mitcheson, J., Perry, M., Stansfeld, P., Sanguinetti, M. C., Witchel, H., and Hancox, J. (2005) Structural determinants for high-affinity block of hERG potassium channels. NoVartis Found Symp. 266, 136150; discussion, 150-138. Kamiya, K., Niwa, R., Mitcheson, J. S., and Sanguinetti, M. C. (2006) Molecular determinants of HERG channel block. Mol. Pharmacol. 69, 1709–1716. Mitcheson, J. S., Chen, J., and Sanguinetti, M. C. (2000) Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate. J. Gen. Physiol. 115, 229–240. Milnes, J. T., Witchel, H. J., Leaney, J. L., Leishman, D. J., and Hancox, J. C. (2006) hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656. Biochem. Biophys. Res. Commun. 351, 273–280. Stork, D., Timin, E. N., Berjukow, S., Huber, C., Hohaus, A., Auer, M., and Hering, S. (2007) State dependent dissociation of HERG channel inhibitors. Br. J. Pharmacol. 151, 1368–1376. Witchel, H. J., Dempsey, C. E., Sessions, R. B., Perry, M., Milnes, J. T, Hancox, J. C., and Mitcheson, J. S. (2004) The low potency, voltagedependent HERG blocker propafenonesMolecular determinants and drug trapping. Mol. Pharmacol. 66, 1201–1212. Yellen, G. (1998) The moving parts of voltage-gated ion channels. Q. ReV. Biophys. 31, 239–295.

Chem. Res. Toxicol., Vol. 21, No. 5, 2008 1009 (29) Webster, S. M., Del Camino, D., Dekker, J. P., and Yellen, G. (2004) Intracellular gate opening in Shaker K+ channels defined by highaffinity metal bridges. Nature 428, 864–868. (30) Labro, A. J., Raes, A. L., Bellens, I., Ottschytsch, N., and Snyders, D. J. (2003) Gating of shaker-type channels requires the flexibility of S6 caused by prolines. J. Biol. Chem. 278, 50724–50731. (31) Mitcheson, J. S., Chen, J., Lin, M., Culberson, C., and Sanguinetti, M. C. (2000) A structural basis for drug-induced long QT syndrome. Proc. Natl. Acad. Sci. U.S.A. 97, 12329–12333. (32) Kamiya, K., Mitcheson, J. S., Yasui, K., Kodama, I., and Sanguinetti, M. C. (2001) Open channel block of HERG K(+) channels by vesnarinone. Mol. Pharmacol. 60, 244–253. (33) Perry, M., de Groot, M. J., Helliwell, R., Leishman, D., TristaniFirouzi, M., Sanguinetti, M. C., and Mitcheson, J. (2004) Structural determinants of HERG channel block by clofilium and ibutilide. Mol. Pharmacol. 66, 240–249. (34) Perry, M., Stansfeld, P. J., Leaney, J., Wood, C., de Groot, M. J., Leishman, D., Sutcliffe, M. J., and Mitcheson, J. S. (2006) Drug binding interactions in the inner cavity of HERG channels: Molecular insights from structure-activity relationships of clofilium and ibutilide analogs. Mol. Pharmacol. 69, 509–519. (35) Ridley, J. M., Milnes, J. T., Duncan, R. S., McPate, M. J., James, A. F., Witchel, H. J., and Hancox, J. C. (2006) Inhibition of the HERG K+ channel by the antifungal drug ketoconazole depends on channel gating and involves the S6 residue F656. FEBS Lett. 580, 1999–2005. (36) Sanchez-Chapula, J. A., Navarro-Polanco, R. A., Culberson, C., Chen, J., and Sanguinetti, M. C. (2002) Molecular determinants of voltagedependent human ether-a-go-go related gene (HERG) K+ channel block. J. Biol. Chem. 277, 23587–23595. (37) Sanchez-Chapula, J. A., Ferrer, T., Navarro-Polanco, R. A., and Sanguinetti, M. C. (2003) Voltage-dependent profile of human ethera-go-go-related gene channel block is influenced by a single residue in the S6 transmembrane domain. Mol. Pharmacol. 63, 1051–1058. (38) Fernandez, D., Ghanta, A., Kauffman, G. W., and Sanguinetti, M. C. (2004) Physicochemical features of the HERG channel drug binding site. J. Biol. Chem. 279, 10120–10127. (39) Ridley, J. M., Milnes, J. T., Witchel, H. J., and Hancox, J. C. (2004) High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: Resistance to mutations of the S6 residues Y652 and F656. Biochem. Biophys. Res. Commun. 325, 883–891. (40) Milnes, J. T., Crociani, O., Arcangeli, A., Hancox, J. C., and Witchel, H. J. (2003) Blockade of HERG potassium currents by fluvoxamine: Incomplete attenuation by S6 mutations at F656 or Y652. Br. J. Pharmacol. 139, 887–898. (41) Dalibalta, S., Leaney, J., de Groot, M. J., Stansfeld, P., and Mitcheson, J. (2008) In Antitargets. Prediction and PreVention of Drug Side Effects (Vaz, R. J., and Klabunde, T., Eds.) pp 89-108, Wiley-VCH, Weinheim. (42) Lees-Miller, J. P., Duan, Y., Teng, G. Q., and Duff, H. J. (2000) Molecular determinant of high-affinity dofetilide binding to HERG1 expressed in Xenopus oocytes: Involvement of S6 sites. Mol. Pharmacol. 57, 367–374. (43) Ficker, E., Jarolimek, W., Kiehn, J., Baumann, A., and Brown, A. M. (1998) Molecular determinants of dofetilide block of HERG K+ channels. Circ. Res. 82, 386–395. (44) Stansfeld, P. J., Gedeck, P., Gosling, M., Cox, B., Mitcheson, J. S., and Sutcliffe, M. J. (2007) Drug block of the hERG potassium channel: Insight from modeling. Proteins 68, 568–580. (45) Herzberg, I. M., Trudeau, M. C., and Robertson, G. A. (1998) Transfer of rapid inactivation and sensitivity to the class III antiarrhythmic drug E-4031 from HERG to M-eag channels. J. Physiol. 511, 3–14. (46) Gessner, G., and Heinemann, S. H. (2003) Inhibition of hEAG1 and hERG1 potassium channels by clofilium and its tertiary analogue LY97241. Br. J. Pharmacol. 138, 161–171. (47) Gessner, G., Zacharias, M., Bechstedt, S., Schonherr, R., and Heinemann, S. H. (2004) Molecular determinants for high-affinity block of human EAG potassium channels by antiarrhythmic agents. Mol. Pharmacol. 65, 1120–1129. (48) Chen, J., Seebohm, G., and Sanguinetti, M. C. (2002) Position of aromatic residues in the S6 domain, not inactivation, dictates cisapride sensitivity of HERG and eag potassium channels. Proc. Natl. Acad. Sci. U.S.A. 99, 12461–12466. (49) Schonherr, R., and Heinemann, S. H. (1996) Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel. J. Physiol. (London) 493, 635–642. (50) Smith, P. L., Baukrowitz, T., and Yellen, G. (1996) The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379, 833–836. (51) Spector, P. S., Curran, M. E., Zou, A., Keating, M. T., and Sanguinetti, M. C. (1996) Fast inactivation causes rectification of the IKr channel. J. Gen. Physiol. 107, 611–619. (52) McPate, M. J., Duncan, R. S., Milnes, J. T., Witchel, H. J., and Hancox, J. C. (2005) The N588K-HERG K+ channel mutation in the ‘short

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(54)

(55)

(56) (57)

(58)

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QT syndrome’: Mechanism of gain-in-function determined at 37 degrees C. Biochem. Biophys. Res. Commun. 334, 441–449. Cordeiro, J. M., Brugada, R., Wu, Y. S., Hong, K., and Dumaine, R. (2005) Modulation of I(Kr) inactivation by mutation N588K in KCNH2: A link to arrhythmogenesis in short QT syndrome. CardioVasc. Res. 67, 498–509. Numaguchi, H., Mullins, F. M., Johnson, J. P., Jr., Johns, D. C., Po, S. S., Yang, I. C., Tomaselli, G. F., and Balser, J. R. (2000) Probing the interaction between inactivation gating and Dd-sotalol block of HERG. Circ. Res. 87, 1012–1018. Ficker, E., Jarolimek, W., and Brown, A. M. (2001) Molecular determinants of inactivation and dofetilide block in ether a-go-go (EAG) channels and EAG-related K+ channels. Mol. Pharmacol. 60, 1343–1348. Cordero-Morales, J. F., Cuello, L. G., and Perozo, E. (2006) Voltagedependent gating at the KcsA selectivity filter. Nat. Struct. Mol. Biol. 13, 319–322. Cordero-Morales, J. F., Cuello, L. G., Zhao, Y., Jogini, V., Cortes, D. M., Roux, B., and Perozo, E. (2006) Molecular determinants of gating at the potassium-channel selectivity filter. Nat. Struct. Mol. Biol. 13, 311–318. Cordero-Morales, J. F., Jogini, V., Lewis, A., Vasquez, V., Cortes, D. M., Roux, B., and Perozo, E. (2007) Molecular driving forces determining potassium channel slow inactivation. Nat. Struct. Mol. Biol. 14, 1062–1069.

Mitcheson (59) Panyi, G., and Deutsch, C. (2007) Probing the cavity of the slow inactivated conformation of shaker potassium channels. J. Gen. Physiol. 129, 403–418. (60) Hille, B. (2001) Classical mechanisms of block. Ion Channels of Excitable Membranes, pp 503-536, Sinauer Associates Inc., Sunderland, MA. (61) De Ponti, F., Poluzzi, E., Montanaro, N., and Ferguson, J. (2000) QTc and psychotropic drugs. Lancet 356, 75–76. (62) Stansfeld, P. J., Sutcliffe, M. J., and Mitcheson, J. S. (2006) Molecular mechanisms for drug interactions with hERG that cause long QT syndrome. Exp. Opin. Drug Metab. Toxicol. 2, 81–94. (63) Sanguinetti, M. C., and Mitcheson, J. S. (2005) Predicting drug-hERG channel interactions that cause acquired long QT syndrome. Trends Pharmacol. Sci. 26, 119–124. (64) Cavalli, A., Poluzzi, E., De Ponti, F., and Recanatini, M. (2002) Toward a pharmacophore for drugs inducing the long QT syndrome: Insights from a CoMFA study of HERG K(+) channel blockers. J. Med. Chem. 45, 3844–3853. (65) Ekins, S., Crumb, W. J., Sarazan, R. D., Wikel, J. H., and Wrighton, S. A. (2002) Three-dimensional quantitative structure-activity relationship for inhibition of human ether-a-go-go-related gene potassium channel. J. Pharmacol. Exp. Ther. 301, 427–434.

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