Ca2+ regulation in neurons by inositol phosphates

通过对IP_3受体（IP_3R）在细胞膜上定位的研究，探讨了在细胞膜上的反应平滑肌细胞和其他细胞以及T细胞我们建立了一种新的方法来分离异源受体群体中的单一类型的肌醇1，4，5-三磷酸受体（IP 3R），环腺苷酸依赖性蛋白激酶（cyclicAMP-dependent protein kinase，PKA）的催化亚基可使~（45）Ca ~（2+）内流到脂蛋白体的速率和程度增加20%。与Snyder的数据相反，它伴随着IP 3受体蛋白的化学计量磷酸化。方法使用免疫细胞化学和分子生物学技术。使用位点特异性抗体的免疫细胞化学研究表明，残基2504-2523位于ER的腔侧。在20个推测的糖基化位点中，只有两个是N-糖基化的候选位点。通过ConA柱层析，Asn-2475和Asn-2503都被糖基化，表明这两个Asn残基位于ER腔侧。这些结果表明，IP 3R穿过膜6次。

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.