Sodium channel selectivity filter regulates antiarrhythmic drug binding (original) (raw)
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Molecular Pharmacology, 2004
Gating properties of Na ϩ channels are the critical determinants for the state-dependent block by class I antiarrhythmic drugs; however, recent site-directed mutagenesis studies have shown that the Na ϩ channel selectivity filter region controls drug access to and dissociation from the binding site. To validate these observations, we have exploited a naturally occurring cardiac Na ϩ channel mutation, S1710L, located next to the putative selectivity filter residue of domain 4, and evaluated the pharmacological properties to mexiletine using whole-cell, patchclamp recordings. Consistent with the large negative shift of steady-state inactivation and the enhanced slow inactivation, the S1710L channel showed greater mexiletine tonic block than wild-type (WT) channel. In contradiction, S1710L showed attenuated use-dependent block by mexiletine and accelerated recovery from block, suggesting that the drug escape though ABBREVIATIONS: WT, wild type.
Circulation Research, 1999
Local anesthetics inhibit Na ϩ channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na ϩ channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX-BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na ϩ channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na ϩ channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can be anchored to the extracellular pore cysteine in cardiac Na ϩ channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors. (Circ Res. 1999;85:88-98.)
Pharmacological modulation of human cardiac Na+ channels
European Journal of Pharmacology: Molecular Pharmacology, 1994
Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na ÷ channels. Na ÷ currents activated near-50 mV with maximum current amplitudes observed at-20 mV. Steady-state inactivation was characterized by a 1111/2 value of-57 + 0.5 mV and a slope factor (k) of 7.3 + 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8/zM. These properties are consistent with those of Na ÷ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na ÷ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na + channels with IC50 values of 521 /zM, 198 /xM, and 41 /zM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na ÷ channels and these drugs compared to other mammalian Na + channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na + channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed and/or removed Na ÷ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the a-subunit is the site of interaction between cardiac Na ÷ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106. Na + channel (human); Oocyte, Class I antiarrhythmic; DPI 201-106
British Journal of Pharmacology, 1999
RSD 921 is a novel, structurally unique, class I Na + channel blocking drug under development as a local anaesthetic agent and possibly for the treatment of cardiac arrhythmias. The eects of RSD 921 on wild-type heart, skeletal muscle, neuronal and non-inactivating IFMQ3 mutant neuronal Na + channels expressed in Xenopus laevis oocytes were examined using a two-electrode voltage clamp. 2 RSD 921 produced similarly potent tonic block of all three wild-type channel isoforms, with EC 50 values between 35 and 47 mM, whereas the EC 50 for block of the IFMQ3 mutant channel was 110+5.5 mM. 3 Block of Na + channels by RSD 921 was concentration and use-dependent, with marked frequency-dependent block of heart channels and mild frequency-dependent block of skeletal muscle, wild-type neuronal and IFMQ3 mutant channels. 4 RSD 921 produced a minimal hyperpolarizing shift in the steady-state voltage-dependence of inactivation of all three wild-type channel isoforms. 5 Open channel block of the IFMQ3 mutant channel was best ®t with a ®rst order blocking scheme with k on equal to 0.11+0.012610 6 M 71 s 71 and k o equal to 12.5+2.5 s 71 , resulting in K D of 117+31 mM. Recovery from open channel block occurred with a time constant of 14+2.7 s 71. 6 These results suggest that RSD 921 preferentially interacts with the open state of the Na + channel, and that the drug may produce potent local anaesthetic or anti-arrhythmic action under conditions of shortened action potentials, such as during anoxia or ischaemia. Keywords: Arylbenzacetamide; RSD 921; class I anti-arrhythmic; Xenopus oocytes; Na + channel Abbreviations: rBIIA, rat brain IIA Na + channel; rH1, rat heart 1 Na + channel; rSkM1 rat skeletal muscle 1 Na + channel
Novel Molecular Determinants in the Pore Region of Sodium Channels Regulate Local Anesthetic Binding
Molecular Pharmacology, 2009
The pore of the Na ϩ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the Nterminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na V 1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5-and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.
Molecular pharmacology, 2001
Membrane-impermeant quaternary amine local anesthetics QX314 and QX222 can access their binding site on the cytoplasmic side of the selectivity filter from the outside in native cardiac Na(+) channels. Mutation of domain IV S6 Ile-1760 of rat brain IIA Na(+) channel or the equivalent (Ile-1575) in the adult rat skeletal muscle isoform (mu 1) creates an artificial access path for QX. We examined the characteristics of mutation of mu 1-I1575 and the resulting QX path. In addition to allowing external QX222 access, I1575A accelerated decay of Na(+) current and shifted steady-state availability by -27 mV. I1575A had negligible effects on inorganic or organic cation selectivity and block by tetrodotoxin (TTX), saxitoxin (STX), or mu-conotoxin (mu-CTX). It exposed a site within the protein that binds membrane-permeant methanethiosulfonate ethylammonium (MTSEA), but not membrane-impermeant methanethiosulfonate ethyltrimethylammonium (MTSET) and methanethiosulfonate ethylsulfonate (MTSES). ...
Proceedings of the National Academy of Sciences, 2000
Membrane-impermeant quaternary derivatives of lidocaine (QX222 and QX314) block cardiac Na ؉ channels when applied from either side of the membrane, but they block neuronal and skeletal muscle channels poorly from the outside. To find the molecular determinants of the cardiac external QX access path, mutations of adult rat skeletal muscle (1) and rat heart (rH1) Na ؉ channels were studied by two-electrode voltage clamp in Xenopus oocytes. Mutating the 1 domain I P-loop Y401, which is the critical residue for isoform differences in tetrodotoxin block, to the heart sequence (Y401C) allowed outside QX222 block, but its mutation to brain type (Y401F) showed little block. 1-Y401C accelerated recovery from block by internal QX222. Block by external QX222 in 1-Y401C was diminished by chemical modification with methanethiosulfonate ethylammonium (MTSEA) to the outer vestibule or by a double mutant (1-Y401C͞F1579A), which altered the putative local anesthetic binding site. The reverse mutation in heart rH1-C374Y reduced outside QX314 block and slowed dissociation of internal QX222. Mutation of 1-C1572 in IVS6 to Thr, the cardiac isoform residue (C1572T), allowed external QX222 block, and accelerated recovery from internal QX222 block, as reported. Blocking efficacy of outside QX222 in 1-Y401C was more than that in 1-C1572T, and the double mutant (1-Y401C͞C1572T) accelerated internal QX recovery more than 1-Y401C or 1-C1572T alone. We conclude that the isoform-specific residue (Tyr͞Phe͞Cys) in the P-loop of domain I plays an important role in drug access as well as in tetrodotoxin binding. Isoform-specific residues in the IP-loop and IVS6 determine outside drug access to an internal binding site.
Cardiovascular …, 2003
The fate of an impulse arising from stimulation is determined by the ability of the wave front to recruit sufficient Na channels from adjacent cells. Previous numerical studies of mutant Na channels revealed both the velocity of a conditioning wave and the recruiting capacity of the front as determinants of the vulnerable period (VP), an interval within which excitation results in unidirectional conduction. Drugs that block excitatory Na channels in a voltage dependent manner, such as antiarrhythmics, abused substances and antidepressants, slow the restoration of Na conductance trailing an action potential and are associated with proarrhythmia and sudden cardiac death. We hypothesized that drug-induced slowing of Na conductance recovery would flatten the Na conductance restoration gradient thereby reducing the recruiting capacity of a front, extending the VP and increasing the probability of unidirectional propagation. Methods: In a cable of ventricular cells, we explored the sensitivity of the VP to voltage-dependent blockade. While varying the unbinding time constant from 100 ms to 5 s, we measured the Na conductance restoration gradient, the liminal length, the refractory period (RP) and the VP. Results: Reducing the rate of drug unbinding flattened the restoration gradient, diminished the recruiting capacity of a premature impulse and extended the liminal length, RP and the VP. The VP was linearly dependent on the drug unbinding time constant. Rapidly unbinding drugs (time constant ,1 s) reduced the liminal length below that of a quiescent cable. Conclusions: Slowing the unbinding rate of voltage-dependent drug block of Na channels extended the RP and the VP. Drugs with unbinding time constants greater than 1 s dramatically increased the probability of unidirectional propagation, reflecting increases in both the RP and the VP. This study provides a new mechanism linking Na channel function, compromised by voltage-dependent Na channel drug block, with proarrhythmic conditions that can lead to sudden cardiac death following premature stimulation.
Journal of Clinical Investigation, 1991
Class I antiarrhythmic drugs inhibit the sodium channel by binding to a drug receptor associated with the channel. In this report we show that in vivo administration of the class I antiarrhythmic drug mexiletine to rats induces sodium channel upregulation in isolated cardiac myocytes. The number of sodium channels was assessed with a radioligand assay using the sodium channel-specific toxin j3HIbatrachotoxinin benzoate (PHIBTXB). The administration of mexiletine to rats induced a dose-dependent increase in PI-HBTXB total specific binding (B.) on isolated cardiac myocytes. Sodium channel numbers were 15±5, 29±9, and 54±4 fmol/105 cells after 3 d treatment with 0, 50 mg/kg per d, and 150 mg/kg per d mexiletine (P < 0.001, analysis of variance). Sodium channel number increased monoexponentially to a steady-state value within 3 d with a half-time of increase of 1.0 d. After cessation of treatment with mexiletine the number of sodium channels returned to normal within 12 d. Finally, treatment with mexiletine altered only sodium channel number, the Kd for I3HjBTXB and the IC5o for mexiletine were not different for myocytes prepared from control and mexiletine-treated rats. (J. Clin. Invest. 1991. 88:375-378.)