Extracellular ions and excitation-contraction coupling in frog twitch muscle fibres

Intracellular calcium transients were recorded from voltage-clamped frog twitch muscle fibres using Arsenazo III. The possible role of extracellular ions in excitation-contraction (e.-c.) coupling was examined using ion substitutions and blocking drugs in the bathing medium. Parameters measured included the Arsenazo response size to a standard depolarizing pulse (5 ms, 0 mV) and the strength-duration curve for threshold Arsenazo signal. Addition of tetrodotoxin (TTX) decreased the response size to small (-30 mV, 5 ms), but not large (+30 mV, 10 ms) depolarizations, probably because of poor voltage clamp of the tubular membrane in the absence of TTX. Clamping TTX-treated fibres with the wave form of a recorded action potential gave an Arsenazo response similar to that elicited by the normal action potential (at 10 degrees C). Complete substitution of sodium (by choline, lithium or Tris) or chloride (by methyl sulphate or maleate) in the bathing solution gave no appreciable changes in the size of the Arsenazo response. Reduction of extracellular free [Ca2+] to low levels using EGTA caused a slight reduction in the calcium signal elicited by the standard depolarization (to 74% after a few hours, and to 62% after 2 days; temperature 5-10 degrees C). The strength-duration curve was unchanged. Arsenazo responses about 75% of the control size could be elicited in high potassium solution (42 mM-K2SO4) by strong (+80 mV, 20 ms) depolarizations, after re-polarizing the fibres to -90 mV for a few minutes. The voltage dependence of activation was shifted to more positive potentials in this solution. Tetraethylammonium (TEA) bromide at a concentration of 20 mM did not alter the Arsenazo signal, whilst 120 mM-TEA reduced the response by 25%. 3,4-diaminopyridine (DAP) reduced the size of the Arsenazo signal at a concentration of 5 mM, and caused spontaneous release of calcium from the sarcoplasmic reticulum (s.r.) in the absence of membrane potential changes. The Arsenazo signal elicited by an action potential was enhanced by 1 mM-DAP, because of prolongation of the action potential, but was depressed by higher concentrations. We conclude that e.-c. coupling does not involve the influx of any external ions into the muscle fibre. If a current flow between the T-tubules and the s.r. is involved in e.-c. coupling, then this is probably carried by an efflux of potassium ions.