The Position of the Fast-Inactivation Gate during Lidocaine Block of Voltage-gated Na 1 Channels

Lidocaine produces voltageand use-dependent inhibition of voltage-gated Na 1 channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na 1 channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel a subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action. key words: local anesthetic • SkM1 • antiarrhythmic • patch clamp • methanethiosulfonate i n t r o d u c t i o n The gating of voltage-sensitive Na 1 channels determines the time course of the rising phase of the action potential and the length of the refractory period in nerve, skeletal muscle, and heart. As a result, Na 1 channels are the targets of several classes of drugs that modulate electrical excitability, including antiarrhythmics, local anesthetics, antimyotonics, and anticonvulsants. Among these, lidocaine and related local anesthetics have received a great deal of experimental attention because of their striking effects on Na 1 channels: they induce a voltage-dependent inhibition of the peak current upon infrequent stimulation (tonic block), and they dramatically slow repriming of sodium channels after depolarizations (use-dependent block), thereby preventing the repetitive discharges that occur in cardiac arrhythmia, epilepsy, and myotonia (Butterworth and Strichartz, 1990). Several experimental findings implicate a role for the Na 1 channel fast inactivation mechanism in generating these effects: depolarization favors local anesthetic binding, many local anesthetics shift the steady state availability ( h ` ) curve in the hyperpolarizing direction (Bean et al., 1983; Hille, 1977), and fast-inactivation– defective Na 1 channels are more resistant to some of the effects of local anesthetics than are normal channels (Cahalan, 1978; Wang et al., 1987; Yeh and Tanguy, 1985). However, a number of questions remain. Is there cooperativity, negative or positive, between lidocaine and the fast-inactivation gate? Does use-dependent block involve an accumulation of fast-inactivated sodium channels? Is there a direct, mutually stabilizing interaction between lidocaine binding and closure of the fast-inactivation gate? Answers to these questions have been difficult to obtain, primarily because gating transitions that occur in drug-bound channels do not involve changes in ionic current, and are thus electrophysiologically silent. Indeed, neither the ionic current nor the gating current provides direct information about the position of the fast-inactivation gate during local anesthetic block. To circumvent this difficulty, we have employed a conformational marker for the position of the fast-inactivation gate, the reactivity of a cysteine substituted for phenylalanine 1304 in the rat adult skeletal muscle sodium channel a subunit with the thiol-modifying reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET). 1 Site 1304 lies in the sodium channel III–IV Address correspondence to Dr. Stephen Cannon, EDR413A, Massachusetts General Hospital, Boston, MA 02214. Fax: 617-726-3926; E-mail: [email protected] 1 Abbreviation used in this paper: MTS-ET, [2-(trimethylammonium)ethyl]methanethiosulfonate. on Jne 3, 2017 D ow nladed fom Published January 1, 1999