Depolarization of skeletal muscle initiates Ca 2+ release from the sarcoplasmic reticulum (SR). Discrete, localized Ca 2+ release events (Ca 2+ """"""""sparks""""""""), which may arise from the coordinated opening of a small 2+ group of SR Ca release channels, or perhaps even from the opening of a single channel, provide a unique window into the operation of Ca 2+ release channels within the normal structural and molecular environment of a functioning skeletal muscle fiber. The global increase in myoplasmic Ca 2+ during fiber depolarization appears to consist of the summation of huge numbers of Ca 2+ sparks initiated during a brief time interval. Our overall goal will be to characterize the properties of these discrete Ca 2+ release events in frog skeletal muscle fibers as a means of determining the cellular and molecular mechanisms controlling the release of Ca 2+ in functioning muscle. We will (1) rigorously compare the properties of voltage activated and spontaneous (ie, ligand gated) Ca2+ sparks in the same muscle fibers, and define conditions under which voltage activated sparks may or may not be associated with """"""""embers,"""""""" (2) determine the properties of Ca 2+ sparks generated in response to single action potentials, (3) investigate modulation of the initiation and generation of """"""""spontaneous"""""""" (ie, ligand gated) Ca 2+ sparks by peptide and protein ligands in permeabilized fibers, (4) optically monitor specific binding of fluorescently labeled peptide and protein modulators of Ca 2+ release to the SR Ca 2+ release channels in permeabilized fibers, (5) investigate the mechanism(s) of initiation and generation of voltage activated Ca2+ sparks, and determine similarities and differences in the mechanisms underlying voltage activated and spontaneous Ca 2+ sparks and (6) investigate possible differences in properties of Ca 2+ sparks in intact compared to cut or permeabilized frog skeletal muscle fibers. Ca 2+ sparks will be monitored by laser scanning confocal fluorescence microscopy, and identified and characterized by detailed computer analysis of the resulting images. The results of these studies should provide further insight into muscle pathological states and to the overall wellness associated with functioning muscle.
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