1521-0111/88/5/866–879$25.00 http://dx.doi.org/10.1124/mol.115.099580 MOLECULAR PHARMACOLOGY Mol Pharmacol 88:866–879, November 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Mechanism of Modification, by Lidocaine, of Fast and Slow Recovery from Inactivation of Voltage-Gated Na1 Channels Vaibhavkumar S. Gawali, Peter Lukacs, Rene Cervenka, Xaver Koenig, Lena Rubi, Karlheinz Hilber, Walter Sandtner, and Hannes Todt Center for Physiology and Pharmacology, Department of Neurophysiology and Neuropharmacology (V.S.G., P.L., R.C., X.K., L.R., K.H., H.T.) and Center for Physiology and Pharmacology (W.S.), Medical University of Vienna, Vienna, Austria Received April 17, 2015; accepted September 9, 2015 Downloaded from ABSTRACT The clinically important suppression of high-frequency dis- mutationsofdomainIVsegment6inrNav1.4 that resulted in charges of excitable cells by local anesthetics (LA) is largely constructs with varying propensities to enter fast- and slow- determined by drug-induced prolongation of the time course of inactivated states. We tested the effect of the LA lidocaine on repriming (recovery from inactivation) of voltage-gated Na1 the time course of recovery from short and long depolarizing channels. This prolongation may result from periodic drug- prepulses, which, under drug-free conditions, recruited mainly molpharm.aspetjournals.org binding to a high-affinity binding site during the action poten- fast- and slow-inactivated states, respectively. Among the tials and subsequent slow dissociation from the site between tested constructs the mutation-induced changes in native slow action potentials (“dissociation hypothesis”). For many drugs it recovery induced by long depolarizations were not correlated has been suggested that the fast inactivated state represents with the respective lidocaine-induced slow recovery after short the high-affinity binding state. Alternatively, LAs may bind with depolarizations. On the other hand, for long depolarizations the high affinity to a native slow-inactivated state, thereby accel- mutation-induced alterations in native slow recovery were erating the development of this state during action potentials significantly correlated with the kinetics of lidocaine-induced (“stabilization hypothesis”). In this case, slow recovery between slow recovery. These results favor the “dissociation hypothe- action potentials occurs from enhanced native slow inactiva- sis” for short depolarizations but the “stabilization hypothesis” tion. To test these two hypotheses we produced serial cysteine for long depolarizations. at ASPET Journals on September 28, 2021 Introduction which underlies their analgesic, anticonvulsive, and antiar- 1 rhythmic efficacy (Macdonald and Kelly, 1993; Pisani et al., Blockers of voltage-gated Na channels are used as local 1995; Weirich and Antoni, 1998; Dupere et al., 1999). anesthetics, antiarrhythmics, antiepileptics, analgesics, and The use-dependent effect of Na1 channel blockers arises in the treatment of specific skeletal muscle diseases. Potential from high-affinity binding to specific states that are periodi- “noncanonical” roles of Na1 channels in nonexcitable cells 1 cally populated during the firing of action potentials. Thus, may open new drug targets for Na channel blockers, such as local anesthetic drugs bind preferentially to activated (Hille, cancer treatment (Black and Waxman, 2013). Recently, there 1977; Yeh and Tanguy, 1985; Wang et al., 1987; McDonald has been an increasing interest in developing blockers with 1 et al., 1989; Vedantham and Cannon, 1999) and/or inactivated high specificity for certain Na channel isoforms. For exam- states (Hille, 1977; Cahalan, 1978; Yeh, 1978; Bean et al., ple, targeting the neuronal isoforms NaV1.7 and NaV1.8 may 1983; Bennett et al., 1995; Balser et al., 1996a). As a result, the lead to the development of potent analgesic drugs with few restoration of excitability between action potentials is pro- side effects (Leffler et al., 2007; Dib-Hajj et al., 2009; Waxman, longed. This latter effect can result from two processes: Drug- 2013; Lampert et al., 2014). Furthermore, targeting the late associated slowing of recovery of excitability could result from: sodium current in the heart may be used in the treatment 1) a slow rate of dissociation of the drug, which slows recovery of arrhythmias, ischemic heart disease, and heart failure from inactivation, (“dissociation-hypothesis”), or 2) an in- (Coppini et al., 2013; Antzelevitch et al., 2014). Functional 1 creased stability of an intrinsic, slow-inactivated state, on specificity of Na channel blockers is thought to arise from the basis of an increased rate of entry into the slow-inactivated their use-dependent effect, which is responsible for the state in the presence of drug (“stabilization hypothesis”; Fig. suppression of high-frequency discharges in excitable cells, 1). These are two fundamentally different mechanisms that have qualitatively similar effects on excitability. With regard This study was funded by the Austrian Science Fund FWF [Grants P210006- B11 and W1232-B11]. to the dissociation hypothesis, drugs are thought to bind with dx.doi.org/10.1124/mol.115.099580. high affinity to a specific state-dependent conformation of the 1 ABBREVIATIONS: DIV, Domain IV; IF, fast inactivation; IFM, intermediate fast inactivation; INa,Na current; IM, intermediate inactivation; IM-MUT, modified intermediate inactivation; IS, slow inactivation; QX-222, N-(2,6-Dimethylphenylcarbamoylmethyl) trimethylammonium chloride; rNaV1.4, rat muscle NaV1.4; S6, segment 6; VGSC, voltage-gated sodium channels. 866 Lidocaine Modifies Fast and Slow Repriming of Nav Channels 867 dissociation from fast-inactivated states and stabilization of slow-inactivated states need not be mutually exclusive, but both mechanisms may operate in concert, as noted by Nuss et al. (2000). This study was designed to systematically investigate drug-induced modification of the time course of recovery from both fast- and slow-inactivated states. There- Fig. 1. Kinetic scheme of proposed state-dependent binding of lidocaine. A three-affinity modulated receptor model in which NI, IF,IM,andIS fore, we applied the local anesthetic lidocaine to channels represent noninactivated states (i.e., pooled open and closed states), fast, exhibiting varying properties of fast and slow inactivation. a a intermediate, and slow-inactivated states, respectively. F and M are To this end we introduced serial cysteine replacements in the the voltage-dependent forward rates into I and I ; b and b are the F M F M segment 6 (S6) of domain IV (DIV) of Na 1.4. This region of respective voltage-dependent backward rates (a9F, a9 M, b9 F, b9 M refer to v the respective rates among lidocaine-bound states). NI-L, IF-L, and IM-L the channel has previously been shown to modulate both fast are the lidocaine-bound NI, IF,andIM states. kon-NI,kon-F and kon-M are and slow inactivation (Chahine et al., 1994; McPhee et al., the on-rates, koff-NI,koff-F,andkoff-M the off-rates for lidocaine binding to 1995, 1998; Chen et al., 1996; Lerche et al., 1997; Sheets NI, IF,andIM. During short depolarizations channels move from NI to IF, during repolarization the transit is from IF to NI (recovery from IF). et al., 1999; Vedantham and Cannon, 2000; Chanda and During long depolarizations channels move from NI to IF and then Bezanilla, 2002; Bosmans et al., 2008; Goldschen-Ohm et al., a b a b further to IM.Sinceboth M and M are smaller than F and F,both 2013) as well as the binding properties of local anesthetic development of and recovery from I require a longer time course than Downloaded from M drugs (Ragsdale et al., 1994, 1996; Yarov-Yarovoy et al., the respective transitions into and out of IF.The“dissociation hypoth- esis” assumes high affinity binding of lidocaine to IF such that during 2002). In addition, we investigated the effect of lidocaine on short depolarizations channels mainly enter the IF-L state. Upon recovery from inactivation in the mutation W1531G. This repolarization channels recover slowly via the small koff-F rate (drug- dissociation), which determines slow dissociation from the high affinity mutation has been shown to generate a pathway for rapid state (IF-L) and via b9 F.The“stabilization hypothesis” assumes high external access and egress of the permanently charged affinity binding of lidocaine to IM (small value for koff-M). Thus, with derivative of lidocaine QX-222 (Lukacs et al., 2014). This lidocaine, even during short depolarizations, channels move from IF-L mutation can be used to study the effects of untrapping of molpharm.aspetjournals.org (low affinity) to IM-L (high affinity). This occurs because, owing to the high affinity of lidocaine to IM-L, the value for koff-M is small (relative to drugs bound to the internal vestibule. We found that most koff-F and koff-NI). Consequently, from detailed balances, a9M has to be probably slow drug dissociation prolongs recovery from brief large, resulting in an acceleration of development of IM-L.Hence,with depolarizations, whereas drug-induced stabilization of slow- the “stabilization hypothesis” recovery from short depolarizations is inactivated states prolongs recovery from long depolariza- prolonged by lidocaine, because channels are moving slowly from IM-L to NI (Chen et al., 2000). The states IFM and IM-MUT are not considered in tions. Furthermore, we found evidence for lidocaine binding this scheme but would be located between IF and IM.