Váradi A, Molnár E, Ashcroft S J
University of Oxford, Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, U.K.
Biochem J. 1996 Mar 1;314 ( Pt 2)(Pt 2):663-9. doi: 10.1042/bj3140663.
Changes in free intracellular Ca2+ concentration regulate insulin secretion from pancreatic beta-cells. The existence of steep Ca2+ gradients within the beta-cell requires the presence of specialized Ca2+ exclusion systems. In this study we have characterized the plasma membrane Ca2+-ATPases (PMCAs) which extrude Ca2+ from the cytoplasm. PMCA isoform- and subtype-specific mRNA expression was investigated in rodent pancreatic alpha- and beta-cell lines, and in human and rat islets of Langerhans using reverse-transcription PCR with primers flanking the calmodulin-binding region of rat PMCA. The expression pattern of PMCA 1 and 2 was conserved in different species and islet-cell types since both rat and human islets of Langerhans and all cell lines tested contained the 1b and 2b forms. PMCA 4 isoform subtypes, however, were expressed in a cell-type-specific manner since beta-cells expressed PMCA 4b only, whereas in islets of Langerhans, which contain alpha, beta, delta and polypeptide-secreting cells, PMCA 4a and 4b were simultaneously present. No evidence was obtained for the expression of PMCA 3. Characterization of the beta-cell Ca2+-pump protein showed that it shared several similarities with the erythrocyte PMCA. It is a P-type ATPase; its phosphorylated intermediate was stabilized by La3+; it reacted with a PMCA-specific antibody; and it was not N-glycosylate. However, the beta-cell PMCA had a higher molecular mass than that of the erythrocyte; this difference could be explained by either predominant translation of the PMCA2 form, which has a molecular mass 3-8 kDa higher than the erythrocyte PMCA 1 and 4 proteins, or by a possible sequence insertion. Thus a unique combination of functionally distinct PMCA isoforms (1b, 2b, 4b) participates in Ca2+ homoeostasis in the beta-cell.
细胞内游离钙离子浓度的变化调节胰腺β细胞的胰岛素分泌。β细胞内陡峭的钙离子梯度的存在需要专门的钙离子排出系统。在本研究中，我们对从细胞质中排出钙离子的质膜钙离子 - ATP酶（PMCAs）进行了表征。使用位于大鼠PMCA钙调蛋白结合区域两侧的引物，通过逆转录PCR研究了啮齿动物胰腺α和β细胞系以及人和大鼠胰岛中PMCA同工型和亚型特异性mRNA的表达。PMCA 1和2的表达模式在不同物种和胰岛细胞类型中是保守的，因为大鼠和人类的胰岛以及所有测试的细胞系都含有1b和2b形式。然而，PMCA 4同工型亚型以细胞类型特异性方式表达，因为β细胞仅表达PMCA 4b，而在含有α、β、δ和多肽分泌细胞的胰岛中，PMCA 4a和4b同时存在。未获得PMCA 3表达的证据。β细胞钙离子泵蛋白的表征表明它与红细胞PMCA有几个相似之处。它是一种P型ATP酶；其磷酸化中间体被La3 +稳定；它与PMCA特异性抗体反应；并且它不是N - 糖基化的。然而，β细胞PMCA的分子量比红细胞的高；这种差异可以通过分子量比红细胞PMCA 1和4蛋白高3 - 8 kDa的PMCA2形式的主要翻译来解释，或者通过可能的序列插入来解释。因此，功能不同的PMCA同工型（1b、2b、4b）的独特组合参与了β细胞中的钙离子稳态。
