Exocytotic Ca2+ sensitivity and the synaptotagmin family

描述（由申请人提供）：本研究计划的目的是了解突触结合蛋白将细胞内Ca 2+浓度增加与活细胞中分泌囊泡的胞吐融合偶联的机制。我们的假设是，对于一个给定的细胞胞吐的Ca 2+敏感性反映了突触结合蛋白亚型的细胞补体的Ca 2+亲和力。针对这一问题，本文提出了三个具体目标：第一，通过阐明突触结合蛋白I-XI的C2结构域抑制神经内分泌PC 12细胞中Ca 2+依赖性胞吐的机制，确定突触结合蛋白-效应子相互作用介导的Ca 2+依赖性分泌。初步数据表明，C2结构域通过破坏内源性突触结合蛋白与脂质磷脂酰肌醇-4，5-二磷酸（PIP 2）或SNARE蛋白SNAP 25和突触融合蛋白的相互作用来抑制。第二，将确定突触结合蛋白家族在广泛的Ca 2+浓度范围内作为Ca 2+传感器的能力。使用生物化学和生物物理学分析，将确定突触结合蛋白I-XI的突触结合蛋白膜和SNARE相互作用的Ca 2+依赖性。最后，将在PC 12细胞中占优势的突触结合蛋白（synaptotagmins）I和IX在神经内分泌细胞中表达，以检测它们是否改变胞吐的Ca 2+敏感性。因此，我们将直接测试我们的假设，即对于给定的细胞胞吐的Ca 2+敏感性反映了细胞的突触结合蛋白亚型的补体的Ca 2+亲和力。更好地了解synaptotagmin家族如何调节神经元胞吐将有助于突触传递的整体机制，以及这个过程如何有助于突触可塑性。了解神经元胞吐作用的调节可能最终为治疗突触传递受损的疾病提供靶点

DESCRIPTION (provided by applicant): The goal of this research plan is to understand the mechanism by which synaptotagmin couples increases in intracellular Ca 2+ concentrations to the exocytotic fusion of secretory vesicles in living cells. Our hypothesis is that the Ca 2+ sensitivity of exocytosis for a given cell reflects the Ca 2+ affinities of the cells complement of synaptotagmin isoforms. To address this question, three Specific Aims are proposed: first, synaptotagmin-effector interactions that mediate Ca 2+ dependent secretion will be identified by elucidating the mechanism of inhibition of Ca 2+ dependent exocytosis in neuroendocrine PC12 cells by C2 domains from synaptotagmins I-XI. Preliminary data indicated that the C2 domains inhibit by disruption of endogenous synaptotagmin interactions with the lipid phosphatidyl-inositol-4,5-bisphosphate (PIP2) or the SNARE proteins SNAP25 and syntaxin. Second, the capacity of the synaptotagmin family to serve as Ca 2+ sensors over a wide range of Ca 2+ concentrations will be determined. Using biochemical and biophysical assay, the Ca 2+ dependency of synaptotagmin membrane and SNARE interactions will be determined for synaptotagmins I-XI. Finally, synaptotagmins identified with Ca 2+ affinities higher or lower than synaptotagmins I and IX, the predominate synaptotagmins in PC12 cells, will be expressed in the neuroendocrine cells to test whether they alter the Ca 2+ sensitivity of exocytosis. Thus, we will directly test our hypothesis that the Ca 2+ sensitivity of exocytosis for a given cell reflects the Ca 2+ affinities of the cell's complement of synaptotagmin isoforms. A better understanding of how the synaptotagmin family regulates neuronal exocytosis will contribute to the overall mechanism of synaptic transmission and how this process contributes to synaptic plasticity. An understanding of the regulation of neuronal exocytosis may ultimately provide targets for treatment of diseases in which synaptic transmission is impaired