A syntaxin 1, Gαo, and N-type calcium channel complex at a presynaptic nerve terminal:: Analysis by quantitative immunocolocalization
A syntaxin 1, Gαo, and N-type calcium channel complex at a presynaptic nerve terminal:: Analysis by quantitative immunocolocalization
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DOI：
10.1523/jneurosci.0346-04.2004
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发表时间：
2004-04-21
影响因子：
5.3
通讯作者：
Stanley, EF
中科院分区：
文献类型：
--
作者：
Li, Q;Lau, A;Stanley, EF
突触前Ca(V)2.2 （n型）钙通道受syntaxin 1和syntaxin 1敏感的Galpha(o) g蛋白途径的相互作用调节。我们利用神经组织裂解物的生化分析和鸡睫状神经节巨大花萼型突触前末端强度相关分析的共定位定量检验，探讨了Ca(V)2.2与syntaxin 1和Galpha(o)的关联。Ca(V)2.2可以通过免疫细胞化学（抗体Ab571）定位在突触前末端释放位点的点孔上，靠近突触囊泡云。Syntaxin 1与Ca(V)2.2共免疫沉淀于鸡脑和鸡睫状神经节，广泛分布于突触前终末膜。总syntaxin 1的一小部分与Ca(V)2.2位点共定位，而大部分与MUNC18-1共定位。无论是三聚体状态还是单体状态，Galpha(o)都不会与Ca(V)2.2、MUNC18-1或syntaxin 1共免疫沉淀。然而，g蛋白在花萼膜上呈现点状染色，其染色强度与Ca通道和syntaxin 1的染色强度同步变化，而与MUNC18-1的染色强度较弱。因此，syntaxin 1似乎是突触前末端两个独立复合物的组成部分，一个较小的复合物位于Ca(V)2.2和Galpha(o)的发射器释放位点，以及位于远离释放位点的MUNC18-1的大簇。这些syntaxin 1蛋白复合物可能在突触前生物学中起着不同的作用。
Presynaptic Ca(V)2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive Galpha(o) G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of Ca(V)2.2 with syntaxin 1 and Galpha(o). Ca(V)2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with Ca(V)2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the Ca(V)2.2 puncta, whereas the bulk colocalized with MUNC18-1. Galpha(o), whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca(V)2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with Ca(V)2.2 and Galpha(o), as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.