The aims of this program are the elucidation of details of the mechanism of the Ca2+ uptake by the (Ca2++Mg2+) ATPase in the sarcoplasmic reticulum (SR), the study of the coupling between the T-tubule system and SR leading to rapid Ca2+ release, and the identification and chaacteriztion of the Ca2+ channel. Specifically, a) answers to a number of unsettled quetions concerning the mechanism of the ATPase reaction will be sought by the combined use of quench flow, rapid filtration and fluorescence probe techniques. Efforts will be aimed at the solution of questions concerning the mechanism by which neighboring subunits of the oligomeric ATPase interact with each other during the Ca2+ pump and Ca2+ release reactions. The possible changes of enzyme conformation during Ca2+ release will also be investigated. b) To solve the problem of the molecular mechanism by which the depolarization signal elicited in the T-tubule membrane is transmitted to the SR leading to rapid Ca2+ release, the in vitro model of excitation-contraction coupling established in this laboratory will be used. Specifically, the effects of experimentally controlled T-tubule membrane potential on Ca2+ release from SR, and key components and key reaction steps involved in T-tubule/Sr coupling will be investigated. The topology of the structural components will be determined by immuno-histochemical localization at the electron microscopic level of antibodies that inhibit the coupling mechanism. c) The Ca2+ release channel of the SR will be identified and characterized through several independent approaches: covalent radio-photoaffinity labeling with channel blockers, localization of the conformational probe whose signal parallels Ca2+ release kinetics, and reconstitution of the Ca2+ channel functions from purified components.
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