Deák Ferenc, Shin Ok-Ho, Kavalali Ege T, Südhof Thomas C
Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, USA.
J Neurosci. 2006 Jun 21;26(25):6668-76. doi: 10.1523/JNEUROSCI.5272-05.2006.
Deletion of synaptobrevin/vesicle-associated membrane protein, the major synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE), severely decreases but does not abolish spontaneous and evoked synaptic vesicle exocytosis. We now show that the closely related R-SNARE protein cellubrevin rescues synaptic transmission in synaptobrevin-deficient neurons but that deletion of both cellubrevin and synaptobrevin does not cause a more severe decrease in exocytosis than deletion of synaptobrevin alone. We then examined the structural requirements for synaptobrevin to function in exocytosis. We found that substituting glutamine for arginine in the zero-layer of the SNARE motif did not significantly impair synaptobrevin-dependent exocytosis, whereas insertion of 12 or 24 residues between the SNARE motif and transmembrane region abolished the ability of synaptobrevin to mediate Ca2+-evoked exocytosis. Surprisingly, however, synaptobrevin with the 12-residue but not the 24-residue insertion restored spontaneous release in synaptobrevin-deficient neurons. Our data suggest that synaptobrevin mediates Ca2+-triggered exocytosis by tight coupling of the SNARE motif to the transmembrane region and hence forcing the membranes into close proximity for fusion. Furthermore, the fusion reactions underlying evoked and spontaneous release differ mechanistically.
突触小泡蛋白/囊泡相关膜蛋白是主要的突触小泡可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（R - SNARE），其缺失会严重降低但不会消除自发和诱发的突触小泡胞吐作用。我们现在表明，密切相关的R - SNARE蛋白细胞ubrevin可挽救突触小泡蛋白缺陷神经元中的突触传递，但细胞ubrevin和突触小泡蛋白两者的缺失并不会比单独缺失突触小泡蛋白导致更严重的胞吐作用降低。然后，我们研究了突触小泡蛋白在胞吐作用中发挥功能的结构要求。我们发现，在SNARE基序的零层用谷氨酰胺替代精氨酸不会显著损害突触小泡蛋白依赖性胞吐作用，而在SNARE基序和跨膜区域之间插入12或24个残基则会消除突触小泡蛋白介导Ca2 +诱发的胞吐作用的能力。然而，令人惊讶的是，插入12个残基而非24个残基的突触小泡蛋白恢复了突触小泡蛋白缺陷神经元中的自发释放。我们的数据表明，突触小泡蛋白通过SNARE基序与跨膜区域的紧密偶联介导Ca2 +触发的胞吐作用，从而迫使膜紧密靠近以进行融合。此外，诱发释放和自发释放背后的融合反应在机制上有所不同。
