An Important Fraction of the Mammalian Skeletal Muscle Chloride Conductance is Located in the Transverse Tubules

Abstract

The actual density of chloride (ClC1) channels in the surface and transverse tubular system (TTS) membranes of mammalian skeletal muscles is still unknown. To investigate this issue, we simultaneously recorded fluorescence signals and chloride currents (ICl) in enzymatically dissociated FDB muscle fibers, stained with the potentiometric indicator di-8-ANEPPS, and voltage-clamped using a 2-microelectrode configuration. The external solution contained (in mM) 150 TEA-Cl, 15 CsMOPS, 2 CaCl2, 0.5 CdCl2, and 200nM TTX. Internally, the fibers were equilibrated with a solution containing 60 CsCl, 40 CsEGTA, 40 CsMOPS, and 5 MgCl2 and voltage-clamped at the chloride equilibrium potential (−20mV). gCl was maximally activated by a pulse to +60mV (150ms) and its voltage-dependence calculated from 9-ACA-sensitive tail currents (measured at the onset of a pulse to −100mV) after 200ms test pulses (−100 to +80mV in amplitude). Boltzmann distributions fitted to the data (n=8) yielded: gClmax=-2.1±0.4 S/F, or 8.1±1.5 mS/cm2; V1/2=73±11 mV and k=24±4 mV. The amplitude (in detaF/F) of di-8-ANEPPS fluorescence transients recorded at the onset of the test pulses were plotted as a function of the pulse amplitudes. In the presence of 9-ACA, the deltaF/F vs. voltage relationship was linear over the entire range of pulse amplitudes explored (slope= −0.124±0.015 /100mV), whereas in the presence of ICl the slope of the linear dependence was less steep. For hyperpolarizing pulses, associated with large instantaneous inward currents, the slope was −0.099±0.03 /100mV; for depolarizing pulses (smaller positive currents) the slope was −0.11±0.025 /100mV. The differential attenuation of the average TTS voltage change in the presence of ICl was predicted by a radial cable model provided that ∼30% of the total gCl was in the TTS. Supported by NIH grants Supported by NIH grants AR07664, and AR054816.