Ca2+ regulation in neurons by inositol phosphates

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With exchanging ideas of localization of IP3 receptor (IP3R) on the plasma membrane, we both detected the reaction on the plasma membrane on caveola structure(by our group) of smooth muscle cells and others and T cell(by Snyder's group).We established a novel method to isolate a single type of inositol 1, 4, 5-trisphosphate receptor(IP3R) among the heterogenous population of receptors, to study the regulatory mechanism of Ca2+ release.Both the rate and extent of 45Ca2+ influx into proteoliposomes were increased 20% after incubation with the catalytic subunit of cyclic AMP-dependent protein kinase(PKA) contrary to Snyder's data It was accompanied by stoichiometric phosphorylation of IP3 receptor protein.Transmembrance topology of IP3R was analyzed by two approaches using immunocytochemical and molecular biological techniques. The immunocytochemical study using the site-specific antibody showed that the residues 2504-2523 are located in the luminal side of the ER. Among the 20 putative glycosylation site only two were the candidates of N-glycosylation. By ConA column chromatography of the site-directed mutant receptors, both Asn-2475 and Asn-2503 were glycosylated, indication the these two Asn residues are located in the ER luminal side. These results suggested that IP3R traverses the membrance six times.