Gladycheva Svetlana E, Ho Chi S, Lee Yue Ying F, Stuenkel Edward L
7804 Medical Sciences II Building, Department of Molecular and Integrative Physiology, The Medical School, University of Michigan, Ann Arbor, MI 48109-0622, USA.
J Physiol. 2004 Aug 1;558(Pt 3):857-71. doi: 10.1113/jphysiol.2004.067249. Epub 2004 Jun 24.
The formation and dissolution of SNARE protein complexes is essential for Ca(2+)-triggered fusion of neurotransmitter-filled vesicles at the presynaptic membrane. Among the pre-synaptic SNARE proteins, the activation of the Q-SNARE syntaxin1A is a critical event for SNARE complex formation. Activation requires syntaxin1A to transit from a munc18-bound non-interacting state to one competent for SNARE binding. The molecular mechanisms that regulate this transition remain unclear. The propensity of syntaxin1A to promote voltage-dependent steady-state inactivation of N-type Ca(2+) channels and accelerate their entry into inactivation was used in a heterologous cell expression system to elucidate regulation of syntaxin1A protein-protein interactions. We report that coexpression of munc18 eliminated the promoting effect of syntaxin1A on inactivation. This effect of munc18 was completely disrupted by coexpression of munc13-1, but not munc13-2 or munc13-3. Also, since expression of munc13-1 with syntaxin1A resulted in an inactivation phenotype identical to that of munc18 with syntaxin1A, the action of munc13-1 on the munc18-syntaxin1A complex was functionally unique and did not result from competitive binding interactions. Furthermore, munc13 expressed with syntaxin1A and munc18 promoted redistribution of a cytosolic SNAP25 mutant to the membrane, a result indicative of syntaxin1A-SNAP25 SNARE pairing. These data demonstrate an important role of munc13 to control the protein-protein interactions of syntaxin1A in vivo, and support munc13 as critical to dissociating syntaxin1A-munc18 complexes and making syntaxin1A available for SNARE interactions.
SNARE蛋白复合物的形成和解离对于Ca(2+)触发的神经递质填充囊泡在突触前膜的融合至关重要。在突触前SNARE蛋白中，Q-SNARE syntaxin1A的激活是SNARE复合物形成的关键事件。激活需要syntaxin1A从与munc18结合的非相互作用状态转变为能够与SNARE结合的状态。调节这种转变的分子机制仍不清楚。在异源细胞表达系统中，利用syntaxin1A促进N型Ca(2+)通道电压依赖性稳态失活并加速其进入失活状态的倾向，来阐明syntaxin1A蛋白质-蛋白质相互作用的调节。我们报告称，munc18的共表达消除了syntaxin1A对失活的促进作用。munc13-1的共表达完全破坏了munc18的这种作用，但munc13-2或munc13-3则没有。此外，由于munc13-1与syntaxin1A的表达导致的失活表型与munc18与syntaxin1A的相同，所以munc13-1对munc18-syntaxin1A复合物的作用在功能上是独特的，并非由竞争性结合相互作用引起。此外，与syntaxin1A和munc18一起表达的munc13促进了胞质SNAP25突变体向膜的重新分布，这一结果表明syntaxin1A-SNAP25 SNARE配对。这些数据证明了munc13在体内控制syntaxin1A蛋白质-蛋白质相互作用中的重要作用，并支持munc13对于解离syntaxin1A-munc18复合物以及使syntaxin1A可用于SNARE相互作用至关重要。
