Cohen R, Atlas D
Department of Biological Chemistry, The Silverman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Neuroscience. 2004;128(4):831-41. doi: 10.1016/j.neuroscience.2004.07.027.
It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca(2+) channels Ca(v)1.2 (Lc-channel) Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II-III domain of the alpha1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca(v)2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca(v)2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca(v)2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca(v)2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca(v)1.2 and Ca(v)2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca(v)2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca(2+)-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca(2+) influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca(2+)-entry. We propose that the distinct kinetics of the Ca(2+)-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.
众所周知， syntaxin 1A、25 kDa的突触体相关蛋白（SNAP - 25）和突触结合蛋白单独或组合起来，可调节电压门控钙通道Ca(v)1.2（L型通道）、Ca(v)2.2（N型）和Ca(v)2.1（P/Q型）的动力学特性。已发现相互作用界面位于通道α1亚基的胞质II - III结构域。在本研究中，我们在注射了BAPTA的非洲爪蟾卵母细胞中证明了人类神经元Ca(v)2.3（R型通道）与syntaxin 1A、SNAP - 25和突触结合蛋白之间存在功能偶联。分别与syntaxin 1A、SNAP - 25或突触结合蛋白组装的Ca(v)2.3的动力学特性不同于与二元复合物syntaxin 1A/SNAP - 25、syntaxin 1A/突触结合蛋白或SNAP - 25/突触结合蛋白相关的Ca(v)2.3。Ca(v)2.3与syntaxin 1A、SNAP - 25和突触结合蛋白一起共表达，产生了一种具有独特动力学特性的通道，类似于由Ca(v)1.2和Ca(v)2.2产生的激发体多蛋白复合物。交换载流离子改变了通道/突触蛋白相互作用的动力学，表明在膜去极化过程中Ca(v)2.3的通透途径与融合装置之间形成了紧密的相互作用。这种假定的偶联可以预测释放位点可能是如何组织的，以便在通道和释放机制之间实现快速通讯。卵母细胞的体内共聚焦成像显示，当通道存在时，GFP - 突触结合蛋白位于质膜上，而在其不存在时则随机分布，这与突触结合蛋白和通道的Ca(2+)非依赖性分子连接一致。在共表达可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNAREs）的卵母细胞的膜上也检测到了突触结合蛋白。非洲爪蟾卵母细胞中的成像研究和蛋白质 - 蛋白质相互作用均表明，通道与突触结合蛋白的连接先于Ca(2+)内流。总之，R型通道似乎在Ca(2+)进入之前就与突触蛋白结合，形成多蛋白激发体复合物。我们提出，通过与激发体复合物中的囊泡和t - SNAREs紧密结合而获得的Ca(2+)通道的独特动力学特性，可能对去极化诱发的递质释放至关重要。
