An elevation of free calcium concentration in the cytoplasmic compartment is an integral component of the mechanism by which cells respond to hormones, growth factors, and certain neurotransmitters. D-myo-inositol 1,4,5-trisphosphate (IP3) is an intracellular messenger mediating the hormonal mobilization of Ca2+ from intracellular stores. This molecule interacts with a specific receptor (IP3R) that has been purified and shown to be a ligand-gated Ca2+ channel. The central theme of this proposal is to study the structure, function, and regulation of IP3 receptors.
The specific aims of the proposal are to investigate: 1) the molecular mechanism of Ca2+ activation. Ca2+ is the principal regulator of IP3R and exerts a biphasic effect on channel function. Acidic amino acids in 2 known calcium-binding regions of the IP3R will be mutated. The mutated receptors will be transiently transfected into COS-7 cells and their functional properties will be assessed using assays that measure the effects of Ca2+ on IP3-mediated 45Ca2+ fluxes, (3H)-IP3 binding and binding to calmodulin (CaM) sepharose. These experiments will test the hypothesis that activation of IP3Rs by Ca2+ is the result of direct Ca2+ binding to high affinity sites in the protein. 2) The molecular mechanism of Ca2+ inhibition. The effect of over-expressing wild type and mutated CaM and the functional consequence of mutation of the known CaM binding site, as well as putative IQ domain calmodulin binding sites will be examined. The experiments will test the hypothesis that the IP3R has multiple CaM binding sites and that CaM binding is responsible for Ca2+ inhibition of the IP3R. 3) Identify residues between transmembrane domains 5 & 6 which play a key role in channel function. Site directed mutations will be made to identify residues that are part of the vestibule of the pore or the pore itself. Ion selectivity of the mutant will be investigated using electrophysiological approaches utilizing patch-clamped nuclei or reconstitution into planar lipid bilayers. These studies are intended to provide insights into the molecular architecture of the conduction pore. 4) Interactions between C-terminal and N-terminal domains of the receptor. Recombinant fusion proteins and in vitro translated transmembrane domains will be used to map the interaction sites on the C-terminal and N-terminal domains. Subsequent mutation of these regions to disrupt the interaction will be used to test the hypothesis that the interaction is fundamental to the mechanism by which ligand binding leads to channel opening. The long term goal of this proposal is to understand how IP3R channels function at a molecular level and to use this knowledge to understand the mechanism by which cells generate the complex spatial and temporal patterns in Ca2+ signaling that underlie a multitude of physiological responses.
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