Detubulation experiments localise delayed rectifier currents to the surface membrane of amphibian skeletal muscle fibres.

Abstract

Ionic currents in intact and detubulated frog sartorius muscle fibres were compared at room temperature using a loose-patch voltage clamp configuration in four experimental groups. The test fibres (i) were detubulated by a previously established osmotic shock protocol that involved the introduction and withdrawal of extracellular glycerol followed by exposure to Ca2+/Mg2+-Ringer solution and cooling. The control fibres were spared osmotic shock and (ii) simply studied in normal Ringer solution, (iii) exposed to 30 min of steady cooling to 9-10 degrees C before electrophysiological study or (iv) exposed to and studied in glycerol-Ringer solution. The presence or absence of detubulation was confirmed for all the experimental groups through assessing for the abolition or otherwise of the delayed after-depolarisation normally associated with action potential propagation into the transverse (T) tubules. All fibre groups showed similar resting potentials (-80 to -90 mV) thus ensuring consistent baseline voltages from which the voltage clamp steps were imposed. The intact muscle fibres in the three control groups (ii)-(iv) spared osmotic shock showed both inward Na+ and delayed rectifier outward (K+) currents. In contrast, patches from detubulated muscle fibres in the test group (i) showed only delayed outward currents, consistent with contrasting contributions to Na+ and K+ currents from regions of membrane affected or spared by the detubulation procedure. Nevertheless, the voltage dependence, maximum steady state amplitudes and timecourses of the delayed outward currents were conserved through all the experimental groups. These findings suggest that the surface as opposed to the tubular membrane contributes the greater part of the delayed rectifier current in amphibian skeletal muscle.