Cytoplasmic ATP-sensing Domains Regulate Gating of Skeletal Muscle ClC-1 Chloride Channels

Abstract

ClC proteins are a family of chloride channels and transporters that are found in a wide variety of prokaryotic and eukaryotic cell types. The mammalian voltage-gated chloride channel ClC-1 is important for controlling the electrical excitability of skeletal muscle. Reduced excitability of muscle cells during metabolic stress can protect cells from metabolic exhaustion and is thought to be a major factor in fatigue. Here we identify a novel mechanism linking excitability to metabolic state by showing that ClC-1 channels are modulated by ATP. The high concentration of ATP in resting muscle effectively inhibits ClC-1 activity by shifting the voltage gating to more positive potentials. ADP and AMP had similar effects to ATP, but IMP had no effect, indicating that the inhibition of ClC-1 would only be relieved under anaerobic conditions such as intense muscle activity or ischemia, when depleted ATP accumulates as IMP. The resulting increase in ClC-1 activity under these conditions would reduce muscle excitability, thus contributing to fatigue. We show further that the modulation by ATP is mediated by cystathionine ␤-synthase-related domains in the cytoplasmic C terminus of ClC-1. This defines a function for these domains as gating-modulatory domains sensitive to intracellular ligands, such as nucleotides, a function that is likely to be conserved in other ClC proteins.Skeletal muscle has a high and variable demand for energy, in the form of ATP, and has elaborate systems to maintain the ATP supply. During intense exercise, however, ATP supply may not keep up with demand, and ATP concentration can decrease rapidly. In fast twitch fibers ATP can drop to below 25% of resting concentration within 25 s (1), a rate of ATP consumption that, if it continued, would deplete all ATP within a further 10 s. As the majority of ATP is consumed by the sarcoplasmic reticulum (SR) 5 Ca 2ϩ -ATPase pumping Ca 2ϩ back into the SR after each Ca 2ϩ -activated contraction (2), complete ATP depletion would lead to a rise in cytoplasmic calcium, rigor, and calcium-dependent damage (3, 4). This does not normally occur because force generation and ATP consumption decrease during exercise, compromising short term function but protecting cells from complete metabolic exhaustion. This process is well known as fatigue, but the factors contributing to fatigue remain controversial. A direct reduction in force generation by the contractile apparatus is thought to be a factor early in fatigue (3, 5), but a significant reduction in ATP consumption only occurs with a reduction in SR Ca 2ϩ release (and consequent reuptake) that occurs late in fatigue, correlating with ATP depletion (3). Indeed, ATP depletion and the concomitant increase in cytoplasmic Mg 2ϩ

Molecular determinants of common gating of a ClC chloride channel

“…As the CBS domains are not directly exposed to the membrane voltage it is most likely that they affect gating allosterically. Mutations in CBS 2 affect ClC-1 common gating to a greater extent than mutations in CBS1 46,51 . Our modelling studies suggest this is because contact between the membrane-embedded channel domain and intracellular CBS domains occurs exclusively in the region of CBS2 (Fig.…”

“…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.…”

“…One possible candidate is ClC-5; however, like ClC-ec1 (refs 56,57), individual ClC-5 monomers appear to competently mediate coupled Cl À /H þ transport without cooperative interactions with the opposing subunit 58 . Like ClC-1 common gating however 46 , ClC-5 function is regulated by intracellular adenosine nucleotides 59 . One possibility is that interactions linking ClC-5 helices G and I may be important for this regulatory mechanism.…”

“…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.…”

Mg 2+ -sensing mechanism of Mg 2+ transporter MgtE probed by molecular dynamics study

“…1 B and D). In fact, CBS domains are present in other transporter proteins and may be involved in the regulation of their transporting activities (14)(15)(16). On the other hand, despite its large movement, the functional role of the N domain remained elusive.…”