C2+ dependence of transverse tubule-mediated calcium release in skinned skeletal muscle fibers

Isometric force and 45Ca efflux from the sarcoplasmic reticulum were measured at 19°C in frog skeletal muscle fibers skinned by microdissection. After Ca2+ loading, application of the ionophores monensin, an Na+(K+)/ H+ exchanger, or gramicidin D, an H+ > K+ > Na+ channel-former, evoked rapid force development and stimulated release of ~30% of the accumulated 45Cawithin 1 min, whereas CCCP (carbonyl cyanide pyruvate p-trichloromethoxyphenylhydrazone), a protonophore, and valinomycin, a neutral, K+- specific ionophore, did not. When monensin was present in all bathing solutions, i.e., before and during Ca2+ loading, subsequent application failed to elicit force development and to stimulate 45Caefflux. 5 min pretreatment of the skinned fibers with 50,µM digitoxin, a permeant glycoside that specifically inhibits the Na+, K+ pump, inhibited monensin and gramicidin D stimulation of 45Caefflux; similar pretreatment with 100 µM ouabain, an impermeant glycoside, was ineffective. Monensin stimulation of 45Ca efflux was abolished by brief pretreatment with 5 mM EGTA, which chelates myofilament-space calcium. These results suggest that: (a) monensin and gramicidin Dstimulate Ca2+ release from the sarcoplasmic reticulum that is mediated by depolarization of the transverse tubules, which seal off after sarcolemma removal and form closed compartments; (b) a transverse tubule membrane potential (myofilament space-negative) is maintained and/or established by the operation of the Na+, K+ pump in the transverse tubule membranes and is sensitive to the permeant inhibitor digitoxin; (c) the transverse tubule-mediated stimulation of 45Caefflux appears to be entirely Ca2+ dependent. © 1986, Rockefeller University Press., All rights reserved.