Inhibition of Skeletal Muscle ClC-1 Chloride Channels by Low Intracellular pH and ATP

Abstract

Skeletal muscle acidosis during exercise has long been thought to be a cause of fatigue, but recent studies have shown that acidosis maintains muscle excitability and opposes fatigue by decreasing the sarcolemmal chloride conductance. ClC-1 is the primary sarcolemmal chloride channel and has a clear role in controlling muscle excitability, but recombinant ClC-1 has been reported to be activated by acidosis. Following our recent finding that intracellular ATP inhibits ClC-1, we investigated here the interaction between pH and ATP regulation of ClC-1. We found that, in the absence of ATP, intracellular acidosis from pH 7.2 to 6.2 inhibited ClC-1 slightly by shifting the voltage dependence of common gating to more positive potentials, similar to the effect of ATP. Importantly, the effects of ATP and acidosis were cooperative, such that ATP greatly potentiated the effect of acidosis. Adenosine had a similar effect to ATP at pH 7.2, but acidosis did not potentiate this effect, indicating that the phosphates of ATP are important for this cooperativity, possibly due to electrostatic interactions with protonatable residues of ClC-1. A protonatable residue identified by molecular modeling, His-847, was found to be critical for both pH and ATP modulation and may be involved in such electrostatic interactions. These findings are now consistent with, and provide a molecular explanation for, acidosis opposing fatigue by decreasing the chloride conductance of skeletal muscle via inhibition of ClC-1. The modulation of ClC-1 by ATP is a key component of this molecular mechanism.

Sarcolemmal-restricted localization of functional ClC-1 channels in mouse skeletal muscle

“…First, these findings validate adequate space clamp uniformity even in naive fibers because ClC-1 channel gating was unaffected by loss of a significant fraction of cell capacitance originating from the T-tubule compartment. Second, previous studies demonstrated that ClC-1 gating is strongly influenced by chloride concentration (Rychkov et al, 1996(Rychkov et al, , 1997Accardi and Pusch, 2000;Chen and Chen, 2001), pH (Rychkov et al, 1996(Rychkov et al, , 1997Accardi and Pusch, 2000;Chen and Chen, 2001;Bennetts et al, 2007), nitric oxide (Lin et al, 2008), and with some controversy, the binding of adenosine derivatives (Bennetts et al, 2005Tseng et al, 2007;Zhang et al, 2008;Zifarelli and Pusch, 2008). Although we did not directly test if these factors modulate native ClC-1 channel activity in FDB fibers, our findings suggest that detubulation by formamide addition and withdrawal does not markedly perturb basal modulatory influences of these factors on ClC-1 gating.…”

Molecular determinants of common gating of a ClC chloride channel

“…Intracellular metabolites such as ATP and NAD þ affect ClC-1 function by shifting the voltage dependence of common gating to more positive potentials and simultaneously reducing the minimum value of common gating curves [45][46][47][48][49][50] . These molecules bind to tandem CBS domains, which form the predominant structural feature of the extensive ClC-1 intracellular carboxy terminus.…”

“…ClC-1 channel activity was assessed using the same methods as detailed elsewhere [45][46][47] . Briefly, channel open probability (P o channel ) and open probability of the common gate (P o common ) were determined from tail-current amplitudes, measured at À 100 mV, following a 300-ms test pulse.…”

Proton-coupled gating in chloride channels

“…External pH was held at 7.5 while the internal pH was varied (5.5, black circles; 6.5, red triangles; 7.5, green squares; 8.5, yellow diamonds). (a,d ) The combined open probability, (b,d ) the slow-gate open probability and (c,e) the fast-gate open probability were determined as described by Bennetts et al 2007. The pipette and bath solutions were composed of (concentrations in mM) 110 NMDG, 110 HCl, 5 MgCl 2 , 1 EGTA, 10 HEPES, pH 7.50, adjusted by NaOH.…”