Skeletal muscle contracts as result of Ca2+ released internally in response to an electrical signal from the surface membrane, but the exact steps of excitation-contraction (EC) coupling are not known. This project is designed to evaluate the adequacy of a single skinned (plasma membrane peeled off) fiber preparation for the study of physiological EC coupling mechanisms, or in other words, skinned fiber Ca2+ release stimulated by via plasma membrane depolarization. The hypotheses to be tested are: 1) Ca2+ release can be elicited by a mechanism inhibited by cardiac-glycosides (CG) and 2) the CG-sensitive component of Ca2+ release is governed by depolarization of plasma membranes (transverse tubules) within the skinned fibers. Rabbit fast and slow twitch fibers and frog fast twitch skeletal muscle fibers will be studied as to their skinned fiber Ca2+ release properties. Ca2+ release will be assayed as isometric tension transients and net 45 Ca2+ release. Stimuli for release are abrupt changes in ionic constituents of the bathing solution thought to bring about depolarization of any sealed-off internal membranes and caffeine. Specific experiments will test whether or not the CG-sensitive component of Ca2+ release is a consequence of depolarization of sealed-off TT's in the skinned fibers and determine the characteristics of CG-sensitive component of Ca2+ release.