Riluzole-induced block of voltage-gated Na⁺ current and activation of BK_Ca channels in cultured differentiated human skeletal muscle cells

Abstract

Riluzole is known to be of therapeutic use in the management of amyotrophic lateral sclerosis. In this study, we investigated the effects of riluzole on ion currents in cultured differentiated human skeletal muscle cells (dHSkMCs). Western blotting revealed the protein expression of α-subunits for both large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel and Na⁺ channel (Na_v1.5) in these cells. Riluzole could reduce the frequency of spontaneous beating in dHSkMCs. In whole-cell configuration, riluzole suppressed voltage-gated Na⁺ current (I_Na) in a concentration-dependent manner with an IC₅₀ value of 2.3 μM. Riluzole (10 μM) also effectively increased Ca²⁺-activated K⁺ current (I_K(Ca)) which could be reversed by iberiotoxin (200 nM) and paxilline (1 μM), but not by apamin (200 nM). In inside-out patches, when applied to the inside of the cell membrane, riluzole (10 μM) increased BK_Ca-channel activity with a decrease in mean closed time. Simulation studies also unraveled that both decreased conductance of I_Na and increased conductance of I_K(Ca) utilized to mimic riluzole actions in skeletal muscle cells could combine to decrease the amplitude of action potentials and increase the repolarization of action potentials. Taken together, inhibition of I_Na and stimulation of BK_Ca-channel activity caused by this drug are partly, if not entirely, responsible for its muscle relaxant actions in clinical setting.

Introduction

Riluzole (2-amino-6-trifuromethoxybenzothiazole) is an anti-glutamate agent which was thought to affect the course of amyotrophic lateral sclerosis (Bensimon et al., 1994, Miller et al., 2003, Cheung et al., 2006). Previous studies demonstrated that this drug possesses anticonvulsant and neuroprotective properties (Couratier et al., 1994, Peluffo et al., 1997, Wahl et al., 1997, Gurney et al., 1998, De Sarro et al., 2000). It was demonstrated to reduce glutamate excitotoxicity by inhibiting glutamate release and/or modifying the postsynaptic glutamate receptor cascade (Martin et al., 1993, Mantz et al., 1994, Rothstein and Kuncl, 1995, De Sarro et al., 2000). In addition, emerging evidence has unraveled that riluzole can inhibit voltage-gated Na⁺ channels (Hebert et al., 1994, Siniscalchi et al., 1997, Song et al., 1997, Yokoo et al., 1998, Zona et al., 1998, Mohammadi et al., 2002). This effect has been proposed to be mainly responsible for the prevention of epilepsy and cellular death caused by this drug (Hebert et al., 1994).

Riluzole has also been reported to activate multiple types of K⁺ channels, including small-conductance Ca²⁺-activated K⁺ channels (Grunnet et al., 2001) and large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels (Wu and Li, 1999). Its activation of BK_Ca channels may also account for the inhibitory action on excitatory neurotransmission (Salkoff et al., 2006, Wu et al., 2006). Thus, the mechanism of action of riluzole on ion channels appears to be complex in multiple types of cells. Although riluzole can inhibit voltage-gated Na⁺ channel and modulate BK_Ca channels in neurons or endocrine cells (Zona et al., 1998, Wu and Li, 1999), the precise mechanism of actions of this drug largely remains unknown in skeletal muscle cells per se.

The large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels, the product of a nearly ubiquitous, alternatively spliced gene (SLO or KCNMA1), have been thought to play a role in regulating excitability of skeletal muscle fibers (Tricarico et al., 2005, Salkoff et al., 2006). They can be synergistically gated open in response to intracellular Ca²⁺ and membrane depolarization (Nimigean and Magleby, 1999, Salkoff et al., 2006, Wu et al., 2006). Skeletal muscle cells are long known to functionally express BK_Ca channels (Moczydlowski and Latorre, 1983, Nimigean and Magleby, 1999), which may mediate responsiveness to relaxant agents and therefore represent a potential target in the treatment of skeletal muscle disorders (Tricarico et al., 2005). Previous studies have demonstrated the ability of riluzole to activate BK_Ca channels in pituitary tumor (GH₃) cells and in pheochromocytoma PC12 cells (Wu and Li, 1999).

Therefore, in this study, we attempted (1) to identify the protein expression of ion channels (i.e., α-subunits for BK_Ca-channel and Na_v1.5) in cultured differentiated human skeletal muscle cells (dHSkMCs); (2) determine the effect of riluzole on macroscopic K⁺ currents in these cells, and (3) to address the issue of whether this drug can have any effects on BK_Ca channels and Na⁺ current (I_Na) in these cells. In our study, we provide substantial evidence that this drug can directly interact with the BK_Ca channel to increase Ca²⁺-activated K⁺ current (I_K(Ca)) and inhibit I_Na in dHSkMCs.