Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat μ1 skeletal muscle sodium channel

Abstract

Mutations that disrupt Na+ channel fast inactivation attenuate lidocaine (lignocaine)‐induced use dependence; however, the pharmacological role of slower inactivation processes remains unclear. In Xenopus oocytes, tryptophan substitution in the outer pore of the rat skeletal muscle channel (μ1‐W402) alters partitioning among fast‐ and slow‐inactivated states. We therefore examined the effects of W402 mutations on lidocaine block. Recovery from inactivation exhibited three kinetic components (IF, fast; IM, intermediate; IS, slow). The effects of W402A and W402S on IF and IS differed, but both mutants (with or without β1 subunit coexpression) decreased the amplitude of IM. In wild‐type channels, lidocaine imposed a delayed recovery component with intermediate kinetics, and use‐dependent block was attenuated in both W402A and W402S. To examine the pharmacological role of IS relative to IM, drug‐exposed β1‐coexpressed channels were subjected to 2 min depolarizations. Lidocaine had no effect on sodium current (INa) after a 1 s hyperpolarization interval that allowed recovery from IM but not IS, suggesting that lidocaine affinity for IS is low. Both W402 mutations reduced occupancy of IM in drug‐free conditions, and also induced resistance to use‐dependent block. We propose that lidocaine‐induced use dependence may involve an allosteric conformational change in the outer pore.

Cardiac-Specific External Paths for Lidocaine, Defined by Isoform-Specific Residues, Accelerate Recovery From Use-Dependent Block

“…Recently, several reports suggested a close relationship between intermediate inactivation (I M ) and lidocaine block of the Na ϩ channel. 20,21 Recovery-time constants for I M were not significantly different among our wild-type and mutant cardiac channels (Table 1). Furthermore, the recovery-time constants for I M did not shorten when pH was raised (57.5Ϯ11.6, 58.2Ϯ4.8, 68.8Ϯ8.1, and 71.4Ϯ10.8 ms for h-WT, h-C373Y, h-T1752V, and h-(C373Y/T1752V), respectively; nϭ5 to 6), unlike those for recovery from lidocaine block.…”

“…These two observations suggest that C1572T mutation in 1, analogous to T1752 in hH1a, opened up a nonpore QX path, which did not overlap with that of the Y401C mutation. Taken together, this raises the interesting possibility that the D4S6 path defined by 20 Amplitudes and time constants in milliseconds are listed (nϭ4 to 6 for all clones). V 1/2 values and Boltzmann slope factors for steady-state inactivation (SSI) with or without lidocaine are listed in bottom half (nϭ5 to 6 for all clones).…”

The Selectivity Filter of the Voltage-gated Sodium Channel Is Involved in Channel Activation

“…If adult rat skeletal muscle (1) Na ϩ channels, heterologously expressed in Xenopus oocytes, are inactivated for Ն20 ms and then repolarized, the channels recover from inactivation with three distinct time constants, implying that there are at least three distinct inactivated states. These time constants are in the order of several ms (fast inactivation), several hundred ms ("intermediate inactivation"), and several thousand ms ("slow inactivation") (1,2). When the channels are inactivated for even longer periods, a component of recovery can be identified with a time constant in the range of 30 -100 s (3)(4)(5)(6).…”

Mechanism of Modification, by Lidocaine, of Fast and Slow Recovery from Inactivation of Voltage-Gated Na+ Channels

“…The time course of recovery following a 10-millisecond prepulse could be fitted by a single exponential equation (eq. 1) with a time constant of 0.57 6 0.03 milliseconds, indicating that all inactivated channels are recovering from a single inactivated state that we refer to as fast-inactivation (I F ; Balser et al, 1996b;Kambouris et al, 1998), defined by time constants of recovery on the order of several milliseconds. If the duration of the conditioning prepulse was prolonged to 50 milliseconds, the time course of recovery could be fitted by the double exponential eq.…”

“…The stabilization hypothesis suggests that drugs bind to native "slow" inactivated states that under drug-free condition require a long time to develop (Khodorov et al, 1976;Zilberter Yu et al, 1991;Balser et al, 1996b;Kambouris et al, 1998;Chen et al, 2000). However, drug-binding to a slow-inactivated state accelerates the time course of development of this state during a depolarization ("stabilization"), thereby increasing the fraction of channels recovering from that state during a subsequent hyperpolarization.…”