Yang S N, Larsson O, Bränström R, Bertorello A M, Leibiger B, Leibiger I B, Moede T, Köhler M, Meister B, Berggren P O
The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10164-9. doi: 10.1073/pnas.96.18.10164.
Interaction of syntaxin 1 with the alpha(1D) subunit of the voltage-gated L type Ca(2+) channel was investigated in the pancreatic beta cell. Coexpression of the enhanced green fluorescent protein-linked alpha(1D) subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the alpha(1D) subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the alpha(1D) subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca(2+) channel activity because there was a faster Ca(2+) current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K(+) or exposure to high Ca(2+). Exchanging Ca(2+) for Ba(2+) abolished the effect of antisyntaxin 1 on both Ca(2+) channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca(2+)-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the alpha(1D) subunit of the L type Ca(2+) channel and the exocytotic machinery in the pancreatic beta cell.
在胰腺β细胞中研究了 syntaxin 1 与电压门控 L 型 Ca(2+)通道的α(1D)亚基之间的相互作用。增强型绿色荧光蛋白连接的α(1D)亚基与增强型蓝色荧光蛋白连接的 syntaxin 1 共表达，结合蛋白质免疫印迹分析和亚细胞分级分离表明，α(1D)亚基和 syntaxin 1 共定位于质膜。此外，α(1D)亚基能被抗 syntaxin 1 的多克隆抗体有效共免疫沉淀。Syntaxin 1 在调节 L 型 Ca(2+)通道活性中也起核心作用，因为与对照相比，用抗 syntaxin 1 处理的细胞中 Ca(2+)电流衰减更快。同时，在用 K(+)去极化或暴露于高 Ca(2+)后，抗 syntaxin 1 显著降低了完整细胞和透化细胞中的胰岛素释放。用 Ba(2+)替代 Ca(2+)消除了抗 syntaxin 1 对 Ca(2+)通道活性和胰岛素胞吐作用的影响。此外，抗 syntaxin 1 对高渗刺激触发的非 Ca(2+)依赖性胰岛素释放没有显著影响。这表明 L 型 Ca(2+)通道的α(1D)亚基与胰腺β细胞中的胞吐机制之间存在结构 - 功能关系。
