García Ricardo A, Godwin Hilary Arnold
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Biophys J. 2004 Apr;86(4):2455-66. doi: 10.1016/S0006-3495(04)74302-7.
Several members of the synaptotagmin (syt) family of vesicle proteins have been proposed to act as Ca2+ sensors on synaptic vesicles. The mechanism by which calcium activates this class of proteins has been the subject of controversy, yet relatively few detailed biophysical studies have been reported on how isoforms other than syt I respond to divalent metal ions. Here, we report a series of studies on the response of syt II to a wide range of metal ions. Analytical ultracentrifugation studies demonstrate that Ca2+ induces protein dimerization upon exposure to 5 mM Ca2+. Whereas Ba2+, Mg2+, or Sr2+ do not potentiate self-association as strongly as Ca2+, Pb2+ triggers self-association of syt II at concentrations as low as 10 microM. Partial proteolysis studies suggest that the various divalent metals cause different changes in the conformation of the protein. The high calcium concentrations required for self-association of syt II suggest that the oligomerized state of this protein is not a critical intermediate in vesicle fusion; however, low-affinity calcium sites on syt II may play a critical role in buffering calcium at the presynaptic active zone. In addition, the high propensity of lead to oligomerize syt II offers a possible molecular explanation for how lead interferes with calcium-evoked neurotransmitter release.
囊泡蛋白的突触结合蛋白（syt）家族的几个成员被认为可作为突触囊泡上的Ca2+传感器。钙激活这类蛋白质的机制一直存在争议，但关于syt I以外的同工型如何响应二价金属离子的详细生物物理研究报道相对较少。在这里，我们报告了一系列关于syt II对多种金属离子响应的研究。分析超速离心研究表明，Ca2+在暴露于5 mM Ca2+时会诱导蛋白质二聚化。而Ba2+、Mg2+或Sr2+增强自我缔合的能力不如Ca2+强，Pb2+在低至10 microM的浓度下就能触发syt II的自我缔合。部分蛋白酶解研究表明，各种二价金属会导致蛋白质构象发生不同变化。syt II自我缔合所需的高钙浓度表明，该蛋白质的寡聚化状态不是囊泡融合的关键中间体；然而，syt II上的低亲和力钙位点可能在突触前活性区缓冲钙方面发挥关键作用。此外，铅使syt II寡聚化的高倾向为铅如何干扰钙诱发的神经递质释放提供了一种可能的分子解释。
