Regulation of ClC-1 and KATP channels in action potential–firing fast-twitch muscle fibers

Abstract

Action potential (AP) excitation requires a transient dominance of depolarizing membrane currents over the repolarizing membrane currents that stabilize the resting membrane potential. Such stabilizing currents, in turn, depend on passive membrane conductance (Gm), which in skeletal muscle fibers covers membrane conductances for K+ (GK) and Cl− (GCl). Myotonic disorders and studies with metabolically poisoned muscle have revealed capacities of GK and GCl to inversely interfere with muscle excitability. However, whether regulation of GK and GCl occur in AP-firing muscle under normal physiological conditions is unknown. This study establishes a technique that allows the determination of GCl and GK with a temporal resolution of seconds in AP-firing muscle fibers. With this approach, we have identified and quantified a biphasic regulation of Gm in active fast-twitch extensor digitorum longus fibers of the rat. Thus, at the onset of AP firing, a reduction in GCl of ∼70% caused Gm to decline by ∼55% in a manner that is well described by a single exponential function characterized by a time constant of ∼200 APs (phase 1). When stimulation was continued beyond ∼1,800 APs, synchronized elevations in GK (∼14-fold) and GCl (∼3-fold) caused Gm to rise sigmoidally to ∼400% of its level before AP firing (phase 2). Phase 2 was often associated with a failure to excite APs. When AP firing was ceased during phase 2, Gm recovered to its level before AP firing in ∼1 min. Experiments with glibenclamide (KATP channel inhibitor) and 9-anthracene carboxylic acid (ClC-1 Cl− channel inhibitor) revealed that the decreased Gm during phase 1 reflected ClC-1 channel inhibition, whereas the massively elevated Gm during phase 2 reflected synchronized openings of ClC-1 and KATP channels. In conclusion, GCl and GK are acutely regulated in AP-firing fast-twitch muscle fibers. Such regulation may contribute to the physiological control of excitability in active muscle.

Sodium channel slow inactivation as a therapeutic target for myotonia congenita

“…9, 10 After blocking muscle contraction with BTS, 5000 action potentials were delivered at 20 Hz in skeletal muscle from ClC adr mice using a protocol similar to one that has been used on wild type skeletal muscle fibers. 11, 12 We examined whether prolonged firing of action potentials led to resolution of myotonia. Myotonia was classified as the continued firing of action potentials following the end of a 60 ms depolarizing pulse.…”

“…31 In wild type rat muscle, it was found that prolonged firing of action potentials triggered a marked increase in membrane conductance. 11, 12 While it appeared that most of the increase in conductance was mediated by ClC-1 choride channels, there was also increased potassium conductance. 11, 12 To measure changes in resting membrane conductance, a 60ms hyperpolarizing pulse was given to measure input resistance.…”

“…BTS was dissolved in DMSO and added to perfusate; maximal concentration of DMSO was 0.1%, less than the concentration of 0.15%, which has been found to not affect resting membrane properties of skeletal muscle. 12 Muscles were stained for 3 minutes with 10μM 4-(4-diethylaminostyrl)-N-methylpyridinium iodide (4-Di-2ASP, Molecular Probes) and imaged with an upright epifluorescence microscope (Leica DMR, Bannockburn, IL) as previously described. 16 For in vitro drug dosing experiments in ClC adr muscles, muscles were prepared as described above and baseline recordings taken.…”

“…11, 12 BTS blocks muscle contraction by inhibiting interaction between myosin and actin, while causing minimal alteration in Ca metabolism or excitability, 9 such that Ca-dependent processes are unperturbed. Using BTS in a mouse model of myotonia congenita, we were able to observe the electrophysiological correlate of the severe stiffness of myotonia at baseline, perform intracellular recordings, and induce warm-up in isolated muscle fibers in the absence of muscle contraction.…”

Novel Insights into the Pathomechanisms of Skeletal Muscle Channelopathies

“…This fits with the clinical observation that in patients with transient weakness, warm-up causes increased strength-quite the opposite of fatigue. Interestingly, investigations into the mechanisms of muscle fatigue by Pedersen et al [47,48] showed that normal muscle can alter its membrane conductances on a time scale of seconds during trains of action potentials. Perhaps this type of phenomenon could be involved in warm-up of myotonic muscle.…”