We propose to study the ionic channels involved directly or indirectly in the process of excitation-contraction coupling in skeletal muscle. Inaccessibility of the transverse tubule (TT) and sarcoplasmic reticulum (SR) membranes have made it difficult or impossible until now to study the conductance pathways contained in these membranes by conventional electrophysiological techniques. In order to study the ionic channels, we will isolate and purify vesicles coming from surface membranes (SM), TT and SR. The membrane origin of the isolated vesicles will be checked by means of several procedures, such as protein and lipid content and composition, enzymatic activities and toxin-binding studies. The presence and density of ionic channels will be measured using specific toxins (TTX and analogues, scorpion toxins, dihydropyridines). We plan to study the single channel properties of SR and TT by incorporating these membranes into plannar bilayers, or in bilayers made on patch-clamp pipettes and/or liposomes. In particular, these methods will be used to study the selectivity, gating, pharmacological and regulation profile of TT calcium-activated potassium channel and of the calcium channel of SR. The SR calcium channel appears to be a possible candidate for the pathway of calcium release in vivo. The long-term goal of the proposal is the detection, characterization and reconstitution of the different conductance pathways contained in muscle membranes. Supporting data demonstrates the feasibility and possible generality of our approach.
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