The secretion of hormones, such as insulin, in response to certain stimuli is known to be associated with an increase of cystosolic free Ca2+. In part, this increase is due to the release of Ca2+ from an internal store but the signal(s) responsible for this release and their mechanism of action are not known. Recent studies have established that phosphatidylinositol-4,5-bisphosphate (a minor phospholipid constituent of the plasma membrane) breaks down in response to a Ca2+ -mobilizing stimulus producing inositol trisphosphate (IP-3), a compound which has the unique ability of rapidly releasing Ca2+ from non-mitochondrial stores. The purpose of this proposal is to study the mechanism of action of this newly identified intracellular messenger as it is expressed in a microsomal preparation obtained from a clonal, insulin-secreting tumor cell. Experiments will be directed towards the following: a) Identifying and characterizing the binding site(s) for IP3 using a radioactively labeled substrate and purified IP-3 -sensitive vesicles. b) Making a detailed analysis of the kinetics of IP-3-mediated Ca2+ release, its temperature dependence, specificity and its interaction with a number of pharmacological agents known to interfere with Ca2+ fluxes. c) Applying voltage-sensitive dyes and other techniques to study the electrophysiology of IP-3-sensitive vesicles. Attempts will be made to distinguish between an electrogenic and electroneutal mechanism. Knowledge of the membrane conductance for Ca2+ in the presence of IP-3 will also be used to distinguish between an ion-channel and a carrier mechanism. d) Attempting to purify and reconstitute the Ca2+ release mechanism. The long-term objective of this proposal is to gain an understanding, at the molecular level, of how IP-3 exerts its physiological effect within cell. The active IP-3 analogue, glycerophosphatidylinositol (4,5)-bisphosphate will be used as the starting point to obtain an affinity column and to synthesize a photoaffinity label of the IP-3 binding site.
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