Closing and Inactivation Potentiate the Cocaethylene Inhibition of Cardiac Sodium Channels by Distinct Mechanisms

Abstract

Cocaethylene, a metabolite of cocaine and alcohol, is a potent inhibitor of the cardiac (Na v 1.5) sodium channel heterologously expressed in Xenopus laevis oocytes. Cocaethylene produces minimal tonic block under resting conditions but causes a potent use-dependent inhibition during repetitive depolarization and a hyperpolarizing shift in the steady-state inactivation. The data are consistent with a state-dependent binding mechanism, which has high affinity for inactivated channels (K I ϭ 17 M) and low affinity for resting channels (K R ϭ 185 M). Mutations of the interdomain D3-D4 linker eliminated rapid inactivation and weakened the cocaethylene inhibition, consistent with an important role for fast inactivation in cocaethylene binding. A rapid component of cocaethylene inhibition was observed in a noninactivating mutant of Nav1.5 that was tightly linked to channel opening and displayed properties consistent with a pore blocking mechanism. Hyperpolarization caused the noninactivating mutant channel to close, trapping cocaethylene and slowing the recovery. Mutation of a conserved isoleucine (I1756C) located near the extracellular end of the D4S6 segment accelerated the recovery of the noninactivating channel, suggesting that this mutation facilitates cocaethylene untrapping, which seems to be the rate-limiting step in the recovery when the channel is closed. This contrasts with the rapidly inactivating channel, where the I1756C mutation did not alter the recovery from cocaethylene inhibition. The data suggest that additional mechanisms, such as more stable cocaethylene binding, may be a more important determinant of recovery kinetics when the channels are inactivated. The data indicate that deactivation and inactivation slow the recovery and potentiate the cocaethylene inhibition of the Nav1.5 channel by distinct mechanisms.

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

“…Two highly conserved aromatic amino acids (F1760 and Y1767) of DIVS6 are known to be critical determinants of drug binding (40). The Y1767C mutation examined in this study is known to weaken the anesthetic inhibition of Na v 1.5 channels, consistent with an important role for this residue in drug binding (36,37). The data show that class I antiarrhythmic drugs (quinidine, mexiletine, lidocaine, and flecainide) are relatively weak inhibitors of the Y1767C-induced persistent Na ϩ current (Fig.…”

“…Y1767 is situated near the cytoplasmic end of the DIVS6 segment and is an important determinant of antiarrhythmic drug binding (36,37). The aromatic side chain at this position appears to be exposed within the aqueous pore, where it is believed to directly interact with bound drugs (1).…”

Access and Binding of Local Anesthetics in the Closed Sodium Channel

“…Mutation I 4i11 A in the muscle and brain Na ϩ channels makes them sensitive to extracellularly applied quaternary LAs (Ragsdale et al, 1994;Wang et al, 1998;Sunami et al, 2001). Furthermore, mutation I 4i11 C facilitates escape of the trapped cocaethylene from Na v 1.5 (O'Leary et al, 2003). Our models explain the effects of these mutations: the bulky Ile 4i11 in the wild type Na v 1.4 prevents LAs to enter the pore, whereas smaller Ala 4i11 enables LAs to pass.…”

“…External QX-222 increased block of the I 4i11 A Na v 1.4 mutant in the presence of the -CTX mutant R13N, suggesting that the outer pore is not the access route for the LA . Furthermore, the mutation I 4i11 C in Na v 1.5 enhanced binding and facilitated escape of cocaethylene from the closed channel (O'Leary et al, 2003).…”

Molecular determinants of state‐dependent block of voltage‐gated sodium channels by pilsicainide

“…The latter is completely impermeant to cell membranes and needs to be applied intracellularly to reach its receptor and inhibit sodium currents. The substitution of an isoleucine residue in the extracellular third of D4S6 (I1760 in rat Nav1.2, I1575 in rat Nav1.4; I1756 in human Nav1.5) with alanine, cysteine or glutamate was shown to create an external access pathway, thereby allowing QX314 to block sodium channels from the outside and facilitate LA drug escape from the closed channel (Ragsdale et al, 1994;Wang et al, 1998;Sunami et al, 2001;O'Leary et al, 2003). In contrast to other isoforms, the cardiac Nav1.5 channel is blocked by external QX314, and replacement of threonine 1765 in rNav1.5 by valine, which is a native residue in rNav1.2 reduced this block (Qu et al, 1995); conversely, a cysteine mutation to threonine at this site in rNav1.4 creates an external access for QX314 (Sunami et al, 2000).…”