Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl− ions. Thus, in resting human muscle, ClC-1 Cl− ion channels account for ∼80% of the membrane conductance, and because active Cl− transport is limited in muscle fibers, the equilibrium potential for Cl− lies close to the resting membrane potential. These conditions—high membrane conductance and passive distribution—enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K+ and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation. The present review summarizes the current knowledge of the physiological factors that control ClC-1 function in active muscle.

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Key pointsr Current methods for determination of the resting membrane conductance in active, action potential-firing muscle fibres are based on the assumptions that the volume and the cytosolic composition of the fibres remain constant during activity.r Here, we present a microelectrode-based method that without such assumptions measures a range of cable parameters including the resting membrane conductance together with action potential characteristics of individual fibres during intermittent action potential firing.r The method can be used to study the role of activity-induced regulation of the resting membrane conductance for action potential excitation and propagation in active muscles.Abstract Recent studies in rat muscle fibres show that repetitive firing of action potentials causes changes in fibre resting membrane conductance (G m ) that reflect regulation of ClC-1 Cl − and K ATP K + ion channels. Methodologically, these findings were obtained by inserting two microelectrodes at close proximity in the same fibres enabling measurements of fibre input resistance (R in ) in between action potential trains. Since the fibre length constant (λ) could not be determined, however, the calculation of G m relied on the assumptions that the specific cytosolic resistivity (R i ) and muscle fibre volume remained constant during the repeated action potential firing. Here we present a three-microelectrode technique that enables determinations of multiple cable parameters in action potential-firing fibres including R in and λ as well as waveform and conduction velocities of fully propagating action potentials. It is shown that in both rat and mouse extensor digitorum longus (EDL) fibres, action potential firing leads to substantial changes in both muscle fibre volume and R i . The analysis also showed, however, that regardless of these changes, rat and mouse EDL fibres both exhibited initial decreases in G m that were eventually followed by a ß3-fold, fully reversible increase in G m after the firing of 1450-1800 action potentials. Using this three-electrode method we further show that the latter rise in G m was closely associated with excitation failures and loss of action potential signal above −20 mV. Abbreviations AP, action potential; BTS, N-benzyl-p-toluene sulphonamide; CSA, muscle fibre cross-sectional area; EDL, extensor digitorum longus; FSA, surface membrane area per unit length of the muscle fibre; g i , intracellular conductivity; g m , membrane conductance per unit length of fibre; G m , membrane conductance per square centimetre of surface membrane; I, injected current; r i , resistance of the intracellular space per unit length; R i , specific cytosolic resistivity; R in , input resistance; r m , membrane resistance per unit length of fibre; SR, sarcoplasmic reticulum; V, magnitude of the membrane voltage response to constant current injections; λ, length constant.

Key pointsr Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)-dependent ClC-1 Cl − channel inhibition in rodent muscle.r While this PKC-dependent ClC-1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle.r Also, the molecular mechanisms underlying the observed PKC-dependent ClC-1 inhibition are unclear.r Here we present the first demonstration of ClC-1 inhibition in active human muscle fibres, and we determine the changes in ClC-1 gating that underlie the PKC-dependent ClC-1 inhibition in active muscle using human ClC-1 expressed in Xenopus oocytes.r This activity-induced ClC-1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity.Abstract Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)-dependent inhibition of ClC-1 Cl − ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC-dependent ClC-1 inhibition are unclear. This study first determined whether PKC-dependent ClC-1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC-1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30-60% reduction of ClC-1 function, which could be completely prevented by PKC inhibition (1 μM GF109203X). The role of the PKC-dependent ClC-1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC-dependent ClC-1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC-1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC-1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC-1. Indeed, there was no effect of PKC activation in C277S mutated ClC-1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC-dependent ClC-1 inhibition via a gating mechanism.

Initiation and propagation of action potentials in muscle fibers is a key element in the transmission of activating motor input from the central nervous system to their contractile apparatus, and maintenance of excitability is therefore paramount for their endurance during work. Here, we review current knowledge about the acute regulation of ClC-1 channels in active muscles and its importance for muscle excitability, function, and fatigue.