CA CHANNEL B SUBUNIT EXCITATION-CONTRACTION COUPLING

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This grant examines the participation of the dihydropyridine receptor (DHPR) beta1a subunit in skeletal muscle excitation-contraction (EC) coupling. In response to depolarization, the DHPR produces a signal that briefly opens ryanodine receptor (RyR) channels leading to the release of stored Ca2+. Considerable progress in the understanding of DHPR-RyR interactions has been made by the availability of mouse models for the expression of DHPRs and RyRs. These are the dysgenic mouse line lacking alpha1S and knockout mice lacking the skeletal muscle RyR1 isoform or lacking the beta1a subunit of the skeletal muscle DHPR. The proposed experiments will use these mutants to identify domains of the beta1a subunit that participate in EC coupling. A C-terminus region of beta1a, unrelated to the BID domain required for binding to alpha1S, will be characterized in detail. This C-terminus domain could bring about a stronger colocalization of DHPRs and RyRs or could be essential for the generation of the signal that opens the RyR. Both possibilities will be tested. To address these questions, we make extensive use of double-null myotubes, (alpha1S/beta1)-null and (beta1/RyR1)-null, generated by mouse breeding. Double-null skeletal muscle cells should permit studies of alpha1S/beta and beta/RyR interactions in expression systems in which the two missing subunits can be expressed and modified. The specific aims of the application are: Aim 1. Establish the role of beta1a in the expression of DHPRs specifically required for EC coupling; Aim 2. Identify molecular domains of alpha1a required for EC coupling; Aim 3. Test functional interactions between beta1a and RyR1 controlling Ca2+ sparks; and Aim 4. Determine whether beta1a triggers a component of the Ca2+ transient. The latter is investigated by expression of alpha1S constructs lacking the II-III loop. The main methods include a) expression of cDNA constructs of alpha1, beta and RyR subunits in single subunit-deficient or in double-null myotubes in culture; b) transgenic overexpression of beta1 constructs; c) macroscopic measurements of Ca2+ currents and charge movements in voltage-clamped myotubes; and d) confocal imaging of Ca2+ transients and Ca2+ sparks. DHPR-RyR interactions are crucial for understanding the molecular basis of EC coupling in normal and diseased states of skeletal muscle.