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The Journal of general physiology

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


PMID 21078869

Abstract

Skeletal muscle fibers exhibit a high resting chloride conductance primarily determined by ClC-1 chloride channels that stabilize the resting membrane potential during repetitive stimulation. Although the importance of ClC-1 channel activity in maintaining normal muscle excitability is well appreciated, the subcellular location of this conductance remains highly controversial. Using a three-pronged multidisciplinary approach, we determined the location of functional ClC-1 channels in adult mouse skeletal muscle. First, formamide-induced detubulation of single flexor digitorum brevis (FDB) muscle fibers from 15-16-day-old mice did not significantly alter macroscopic ClC-1 current magnitude (at -140 mV; -39.0 +/- 4.5 and -42.3 +/- 5.0 nA, respectively), deactivation kinetics, or voltage dependence of channel activation (V(1/2) was -61.0 +/- 1.7 and -64.5 +/- 2.8 mV; k was 20.5 ± 0.8 and 22.8 +/- 1.2 mV, respectively), despite a 33% reduction in cell capacitance (from 465 +/- 36 to 312 +/- 23 pF). In paired whole cell voltage clamp experiments, where ClC-1 activity was measured before and after detubulation in the same fiber, no reduction in ClC-1 activity was observed, despite an approximately 40 and 60% reduction in membrane capacitance in FDB fibers from 15-16-day-old and adult mice, respectively. Second, using immunofluorescence and confocal microscopy, native ClC-1 channels in adult mouse FDB fibers were localized within the sarcolemma, 90 degrees out of phase with double rows of dihydropyridine receptor immunostaining of the T-tubule system. Third, adenoviral-mediated expression of green fluorescent protein-tagged ClC-1 channels in adult skeletal muscle of a mouse model of myotonic dystrophy type 1 resulted in a significant reduction in myotonia and localization of channels to the sarcolemma. Collectively, these results demonstrate that the majority of functional ClC-1 channels localize to the sarcolemma and provide essential insight into the basis of myofiber excitability in normal and diseased skeletal muscle.