Atomic determinants of state-dependent block of sodium channels by charged local anesthetics and benzocaine

Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ont., Canada L8N 3Z5.
FEBS Letters (Impact Factor: 3.34). 12/2006; 580(26):6027-32. DOI: 10.1016/j.febslet.2006.10.035
Source: PubMed

ABSTRACT Molecular modeling predicts that a local anesthetic (LA) lidocaine binds to the resting and open Na(v)1.5 in different modes, interacting with LA-sensing residues known from experiments. Besides the major pathway via the open activation gate, LAs can reach the inner pore via a "sidewalk" between D3S6, D4S6, and D3P. The ammonium group of a cationic LA binds in the focus of the pore-helices macrodipoles, which also stabilize a Na(+) ion chelated by two benzocaine molecules. The LA's cationic group and a Na(+) ion in the selectivity filter repel each other suggesting that the Na(+) depletion upon slow inactivation would stabilize a LA, while a LA would stabilize slow-inactivated states.

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    • "In homology models of sodium and calcium channels, corresponding interfaces between domains III and IV contain residues whose mutations affect access and binding of different ligands (Zhorov and Tikhonov, 2004; Bruhova et al., 2008; Tikhonov and Zhorov, 2008; Cheng et al., 2009). Extracellularly applied permanently charged compounds, which cannot permeate the membrane , were proposed to reach their binding sites within the inner pore through the III/IV domain interface (Tikhonov et al., 2006; Bruhova et al., 2008; Tikhonov and Zhorov, 2008). Some ligands of the Kv1.5 channel were also proposed to reach their binding site via the intersubunit interface (Strutz-Seebohm et al., 2007). "
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    ABSTRACT: hKv1.3 channels in lymphocytes are targets for the chemotherapy treatment of autoimmune diseases. Phenylalkylamines block Kv1.3 channels by poorly understood mechanisms. In the inactivation-reduced mutant H399T, the second mutation A413C in S6 substantially decreases the potency of phenylalkylamines with a para-methoxy group at the phenylethylamine end, whereas potency of phenylalkylamines lacking this group is less affected. Intriguingly, completely demethoxylated emopamil blocks mutant H399T/A413C with a 2:1 stoichiometry. Here, we generated a triple mutant, H399T/C412A/A413C, and found that its emopamil-binding properties are similar to those of the double mutant. These data rule out disulfide bonding Cys412-Cys413, which would substantially deform the inner helix, suggest a clash of Cys413 with the para-methoxy group, and provide a distance constraint to dock phenylalkylamines in a Kv1.2-based homology model. Monte Carlo minimizations predict that the verapamil ammonium group donates an H-bond to the backbone carbonyl of Thr391 at the P-loop turn, the pentanenitrilephenyl moiety occludes the pore, whereas the phenylethylamine meta- and para-methoxy substituents approach, respectively, the side chains of Met390 and Ala413. In the double-mutant model, the Cys413 side chains accept H-bonds from two emopamil molecules whose phenyl rings fit in the hydrophobic intersubunit interfaces, whereas the pentanenitrilephenyl moieties occlude the pore. Because these interfaces are unattractive for a methoxylated phenyl ring, the ammonium group of respective phenylalkylamines cannot approach the Cys413 side chain and binds at the focus of P-helices, whereas the para-methoxy group clashes with Cys413. Our study proposes an atomistic mechanism of Kv1.3 block by phenylalkylamines and highlights the intra- and intersubunit interfaces as ligand binding loci.
    Molecular pharmacology 03/2011; 79(4):681-91. DOI:10.1124/mol.110.068031 · 4.12 Impact Factor
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    • "This concept allowed us to recently propose structural models that explain long-known effects of metal ions on L-type Ca 2ϩ channel ligands (Tikhonov and Zhorov, 2008; Cheng et al., 2009; Tikhonov and Zhorov, 2009). We also explained previously the observations that the cationic lidocaine and the uncharged benzocaine block Na ϩ channels with Hill coefficients of 1 and 2, respectively, by a model in which two benzocaine molecules coordinate an Na ϩ ion in the permeation pathway (Tikhonov et al., 2006). Based on the above-described experiments and the fact that the uncharged PAP-1 inhibits Kv1.3 with a Hill coefficient of 2 (Schmitz et al., 2005), we hypothesized that Kv1.3 is blocked by a tripartite complex consisting of two PAP-1 molecules and a K ϩ ion. "
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    ABSTRACT: Voltage-gated potassium channels (Kv) are targets for drugs of large chemical diversity. Although hydrophobic cations block Kv channels with Hill coefficients of 1, uncharged electron-rich (cationophilic) molecules often display Hill coefficients of 2. The mechanism of the latter block is unknown. Using a combination of computational and experimental approaches, we mapped the receptor for the immunosuppressant PAP-1 (5-(4-phenoxybutoxy)psoralen), a high-affinity blocker of Kv1.3 channels in lymphocytes. Ligand-docking using Monte Carlo minimizations suggested a model in which two cationophilic PAP-1 molecules coordinate a K(+) ion in the pore with their coumarin moieties, whereas the hydrophobic phenoxyalkoxy side chains extend into the intrasubunit interfaces between helices S5 and S6. We tested the model by generating 58 point mutants involving residues in and around the predicted receptor and then determined their biophysical properties and sensitivity to PAP-1 by whole-cell patch-clamp. The model correctly predicted the key PAP-1-sensing residues in the outer helix, the P-loop, and the inner helix and explained the Hill coefficient of 2 by demonstrating that the Kv1.3 pore can accommodate two or even four PAP-1 molecules. The model further explained the voltage-dependence of block by PAP-1 and its thousand-fold selectivity for Kv1.3 over non-Kv1 channels. The 23- to 125-fold selectivity of PAP-1 for Kv1.3 over other Kv1 channels is probably due to its preferential affinity to the C-type inactivated state, in which cessation of K(+) flux stabilizes the tripartite PAP-1:K(+):PAP-1 complex in the pore. Our study provides a new concept for potassium channel block by cationophilic ligands.
    Molecular pharmacology 10/2010; 78(4):588-99. DOI:10.1124/mol.110.064014 · 4.12 Impact Factor
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    • "We have shown previously that the block of sodium channels by 2,6-dimethylphenol, a structural analogue of the aromatic alcohol and anaesthetic propofol on the one hand and the aromatic tail of lidocaine-like local anaesthetics on the other, mimics important features of sodium channel blockade by its parent compounds (Haeseler et al., 2002). Recent molecular modelling studies involving several analogues of the local anaesthetic benzocaine have raised the hypothesis that the substituted benzene ring containing a functionally active group capable of binding a Na þ ion (and thus mimicking the ammonium group of a charged local anaesthetic) constitutes the effective principle of local anaesthetic molecules because it determines their interaction with amino-acid residues critical for local anaesthetic binding in a hydrophobic cavity on the S6 segment of domain 4 in voltage-operated human skeletal muscle sodium channels (Na V 1.4) (Tikhonov et al., 2006; Godwin et al., 2005). Thus, it seems likely that phenol derivatives and benzocaine analogues act via a common local anaesthetic receptor site. "
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    ABSTRACT: Voltage-operated sodium channels constitute major target sites for local anaesthetic-like action. The clinical use of local anaesthetics is still limited by severe side effects, in particular, arrhythmias and convulsions. These side effects render the search for new local anaesthetics a matter of high interest. We have investigated the effects of three halogenated structural analogues of propofol on voltage-operated human skeletal muscle sodium channels (Na(V)1.4) and the effect of one compound (4-chloropropofol) on neuronal sodium channels (Na(V)1.2) heterologously expressed in human embryonic kidney cell line 293. 4-Iodo-, 4-bromo- and 4-chloropropofol reversibly suppressed depolarization-induced whole-cell sodium inward currents with high potency. The IC(50) for block of resting channels at -150 mV was 2.3, 3.9 and 11.3 microM in Na(V)1.4, respectively, and 29.2 microM for 4-chloropropofol in Na(V)1.2. Membrane depolarization inducing inactivation strongly increased the blocking potency of all compounds. Estimated affinities for the fast-inactivated channel state were 81 nM, 312 nM and 227 nM for 4-iodopropofol, 4-bromopropofol and 4-chloropropofol in Na(V)1.4, and 450 nM for 4-chloropropofol in Na(V)1.2. Recovery from fast inactivation was prolonged in the presence of drug leading to an accumulation of block during repetitive stimulation at high frequencies (100 Hz). Halogenated propofol analogues constitute a novel class of sodium channel-blocking drugs possessing almost 100-fold higher potency compared with the local anaesthetic and anti-arrhythmic drug lidocaine. Preferential drug binding to inactivated channel states suggests that halogenated propofol analogues might be especially effective in suppressing ectopic discharges in a variety of pathological conditions.
    British Journal of Pharmacology 07/2008; 155(2):265-75. DOI:10.1038/bjp.2008.255 · 4.99 Impact Factor
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