Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis
Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis
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DOI：
10.1093/emboj/cdf404
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发表时间：
2002-08-01
期刊：
影响因子：
11.4
通讯作者：
Seagar, M
中科院分区：
文献类型：
--
作者：
Quetglas, S;Iborra, C;Seagar, M
神经递质释放涉及异源三聚体 SNARE 复合物的组装，该复合物由囊泡蛋白突触短蛋白 (VAMP 2) 和两个质膜伴侣（突触蛋白 1 和 SNAP-25）组成。钙流入被认为通过与 SNARE 复合体成分相关的 Ca2+ 结合蛋白来控制这一过程。 Ca2+/钙调蛋白或磷脂以互斥的方式与 VAMP (VAMP(77-90)) 的 C 末端结构域结合，并通过等离振子共振光谱法鉴定了涉及的残基。通过碳纤维电流分析法监测，显微注射野生型 VAMP(77-90)（而非突变肽）可抑制嗜铬细胞中儿茶酚胺的释放。在透化细胞测定中，将 PC12 嗜铬细胞瘤细胞与不可逆钙调蛋白拮抗剂 ophiobolin A 预孵育可抑制 Ca2+ 依赖性人生长激素的释放。用破伤风毒素轻链（TeNT）处理透化细胞也抑制了分泌。在 TeNT 存在的情况下，通过转染 TeNT 抗性 (Q(76)V, F77W) VAMP 可以恢复胞吐作用，但 VAMP(77-90) 中的其他靶向突变消除了其拯救释放的能力。因此，VAMP 2 的钙调蛋白和磷脂结合域是 Ca2+ 依赖性胞吐作用所必需的，可能是为了调节 SNARE 复合物的组装。
Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca2+-binding proteins that associate with components of the SNARE complex. Ca2+/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP(77-90)), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP(77-90), but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca2+-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q(76)V, F77W) VAMP, but additional targeted mutations in VAMP(77-90) abolished its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca2+-dependent exocytosis, possibly to regulate SNARE complex assembly.