Sørensen Jakob B, Matti Ulf, Wei Shun-Hui, Nehring Ralf B, Voets Thomas, Ashery Uri, Binz Thomas, Neher Erwin, Rettig Jens
Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany.
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1627-32. doi: 10.1073/pnas.251673298.
Synchronous neurotransmission depends on the tight coupling between Ca(2+) influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.
同步神经传递依赖于钙离子内流与充满神经递质的囊泡与质膜融合之间的紧密偶联。囊泡可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白突触小泡蛋白2以及质膜SNARE蛋白 syntaxin 1和25 kDa的突触体相关蛋白（SNAP - 25）对于钙触发的胞吐作用至关重要。然而，钙触发与SNARE功能之间的联系仍然难以捉摸。在这里，我们描述了由SNAP - 25和突触小泡蛋白2的酸性和亲水残基形成的SNARE复合物表面两个位点的突变，在神经元SNARE复合物晶体结构中发现这些位点可配位二价阳离子。通过降低SNAP - 25中该位点的净电荷，我们鉴定出一种突变，当在嗜铬细胞中过表达时，该突变会干扰钙触发的胞吐作用。通过从钙笼中光释放钙离子引发胞吐作用，并使用毫秒时间分辨率的膜片钳电容测量进行评估。我们提出了一种监测释放位点胞吐速率对钙浓度依赖性的方法，并证明该突变降低了这种关系的陡度，表明胞吐作用之前连续钙结合步骤的数量减少了一个。我们得出结论，SNARE复合物直接与钙触发的胞吐作用相关联，很可能是与辅助蛋白形成复合物。
