Ryanodine receptors are high conductance, intracellular calcium channels embedded in the sarcoplasmic reticulum of muscle cells. Muscle contraction is elicited with ryanodine receptors open, thereby allowing calcium stored in the sarcoplasmic reticulum to rapidly enter the cytoplasm. The resulting increase in cytoplasmic calcium concentrations directly activates muscle contraction. Ryanodine receptors play a central role in excitation-contraction coupling, the signal transduction pathway by which depolarization of the muscle cell plasma membrane leads to contraction of the myofilament lattice. We will investigate the roles that C. elegans ryanodine receptors play in regulating muscle contraction. We will use a genetics-centered multidisciplinary approach to analyze unc-68, the only ryanodine receptor gene present in C. elegans. We will perform structure/function analysis of UNC-68 by engineering site directed mutations that affect putative UNC-68 regulatory sites, and determining the effect that such mutations have on UNC-68 function both in vivo and in vitro. We will genetically investigate excitation-contraction coupling in C. elegans using a variety of approaches. We will isolate mutations that phenotypically suppress the contractile defects of unc-68 mutants. We will identify additional genes involved in excitation-contraction coupling by isolating mutants which, like unc-68, are resistant to the paralyzing effects of ryanodine. We will investigate genetic interactions between unc-68 and existing mutations known or thought to affect excitation- contraction coupling. We will characterize molecularly and genetically C. elegans homologs of certain vertebrate genes that modulate ryanodine receptor activity. Our approach combined genetic manipulation of C. elegans with cell biological descriptions and biophysical characterization of the affected processes. Our long-term goal is to understand the regulation of muscle contraction in C. elegans. his work is important in understanding the genetic basis of Malignant Hyperthermia and Central Core Disease, human disorders caused by inherited mutations of skeletal muscle ryanodine receptors.
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