The two principal intracellular calcium release channels involved in calcium mobilization inside cells are the ryanodine and inositol 1,4,5- trisphosphate (InsP3) receptors. They exhibit a high degree of sequence homology. Ca2+ release mediated by the InsP3 receptor is known to be unusual in that it can be triggered several times in succession by elevating the InsP3 concentration in small increments. Unlike the case with conventional receptor desensitization, these receptors can be re- activated by further increases in ligand concentration without removal of the ligand in between. Such release was originally referred to as """"""""quantal"""""""" because it manifested features like that of a release of Ca2+ from a heterogeneous population of vesicles each releasing their Ca2+ in an all-or-none fashion, but other hypotheses have since been proposed to explain the phenomenon. While investigated extensively since 1989, no clear explanation has emerged as to the underlying mechanism. In 1993 an apparently similar process was reported for the muscle ryanodine receptor. The present proposal will systematically test hypotheses directed at answering the following questions: I.) Is Ca2+ involved on either side of the membrane? II.) Is another protein involved? III.) Is the behavior an inherent property of the intracellular Ca2+ release channel itself? IV.) Is the phenomenon manifest at the cellular level in the cardiovascular system, and how might it be accelerated in situ? Both InsP3 and ryanodine receptors will be tested, especially since recent findings with ryanodine receptors cast doubt on certain hypotheses originally proposed for the InsP3 receptor. The preparations that will be used for the majority of these studies include cardiac microsomes and the most highly enriched source of native membranes known for each receptor, skeletal muscle SR terminal cisternae for the ryanodine receptor, and cerebellar microsomes for the InsP3 receptor. Experiments will utilize pharmacologic manipulation, spectrophotometry, isotope flux and planar lipid bilayer single channel recording techniques. A computer model will be developed to generate a coherent explanation of how this unusual behavior takes place, and its in situ significance will be assessed with intracellular [Ca2+] measurements on cardiovascular tissue, ventricular myocytes and mesenteric resistance artery. These studies should reveal the underlying molecular mechanism for this newly discovered process that regulates muscle contraction, signal transduction within the central nervous system and processes controlled by Ca2+ oscillations in both excitable and non-excitable cells. Alterations in this process are likely to contribute to various pathologic states, particularly episodes of malignant hyperthermia and arrhythmias brought about by intracellular Ca2+ overload.
