The transmitter release-site CaV2.2 channel cluster is linked to an endocytosis coat protein complex
The transmitter release-site CaV2.2 channel cluster is linked to an endocytosis coat protein complex
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DOI：
10.1111/j.1460-9568.2007.05681.x
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发表时间：
2007-08-01
影响因子：
3.4
通讯作者：
Stanley, Elise F.
中科院分区：
文献类型：
--
作者：
Khanna, Rajesh;Li, Qi;Stanley, Elise F.
突触囊泡（SV）通过Ca 2+通过附近附着的CaV 2.2通道内流而被触发与突触前递质释放位点（TRS）核心处的表面膜融合[参见随附论文：卡纳等人（2007）Eur.神经科学杂志，26，547-559]，然后通过内吞作用回收。在这项研究中，我们测试的假设，TRS核心是连接到一个内吞相关的蛋白质复合物。这是测试通过免疫染色分析的鸡睫状神经节萼突触前末端和突触体裂解物的生化分析，使用CaV2.2作为标记的TRS。我们注意到CaV2.2簇与发射器释放面的重链（H）-网格蛋白斑块相邻。定量共免疫染色分析（伊卡/ICQ方法）表明，释放面CaV2.2染色与AP 180和interstin内吞衔接蛋白具有较强的协方差性，与H-或轻链（L）-网格蛋白和发动蛋白外壳蛋白具有中等协方差性，与多分子复合物一致。这得到了这些蛋白质与来自脑突触体裂解物的CaV2.2的共沉淀的支持。有趣的是，通道既不共定位，也不共沉淀与内吞作用的货物捕获适配器AP 2，即使这种蛋白质共定位和共沉淀与H-网格蛋白。通过暴露于高NaCl（类似于1 M）的免疫沉淀的CaV2.2复合物的部分恢复分析表明，AP 180和S-交叉蛋白衔接子与CaV2.2紧密结合，而L-交叉蛋白、H-和L-网格蛋白和发动蛋白形成较不紧密连接的亚复合物。我们的研究结果是一致的两个不同的网格蛋白内吞复合物：一个AP 2含，远程，非TRS复杂和一个专门的，AP 2缺乏，TRS相关的子复合物通过分子桥连接。该亚复合体最可能的作用是促进SV释放后的恢复。
Synaptic vesicles (SVs) are triggered to fuse with the surface membrane at the presynaptic transmitter release site (TRSs) core by Ca2+ influx through nearby attached CaV2.2 channels [see accompanying paper: Khanna et al. (2007) Eur. J. Neurosci., 26, 547-559] and are then recovered by endocytosis. In this study we test the hypothesis that the TRS core is linked to an endocytosis-related protein complex. This was tested by immunostaining analysis of the chick ciliary ganglion calyx presynaptic terminal and biochemical analysis of synaptosome lysate, using CaV2.2 as a marker for the TRS. We noted that CaV2.2 clusters abut heavy-chain (H)-clathrin patches at the transmitter release face. Quantitative coimmunostaining analysis (ICA/ICQ method) demonstrated a strong covariance of release-face CaV2.2 staining with that for the AP180 and intersectin endocytosis adaptor proteins, and a moderate covariance with H- or light-chain (L)-clathrin and dynamin coat proteins, consistent with a multimolecular complex. This was supported by coprecipitation of these proteins with CaV2.2 from brain synaptosome lysate. Interestingly, the channel neither colocalized nor coprecipitated with the endocytosis cargo-capturing adaptor AP2, even though this protein both colocalized and coprecipitated with H-clathrin. Fractional recovery analysis of the immunoprecipitated CaV2.2 complex by exposure to high NaCl (similar to 1 M) indicated that AP180 and S-intersectin adaptors are tightly bound to CaV2.2 while L-intersectin, H- and L-clathrin and dynamin form a less tightly linked subcomplex. Our results are consistent with two distinct clathrin endocytosis complexes: an AP2-containing, remote, non-TRS complex and a specialised, AP2-lacking, TRS-associated subcomplex linked via a molecular bridge. The most probable role of this subcomplex is to facilitate SV recovery after transmitter release.