Exocytotic Ca2+ sensitivity and the synaptotagmin family

描述(申请人提供)：这项研究计划的目标是了解突触素偶联增加细胞内钙离子浓度以促进活细胞内分泌囊泡的胞外融合的机制。我们的假设是，特定细胞胞吐作用的钙敏感性反映了细胞内突触素亚型互补的钙亲和力。为了解决这个问题，提出了三个具体的目标：第一，通过阐明突触素I-XI的C2结构域抑制神经内分泌细胞钙依赖性胞吐的机制，鉴定介导钙依赖性分泌的突触素-效应器相互作用。初步数据表明，C2结构域通过破坏内源性突触素与磷脂酰肌醇-4，5-二磷酸(PIP2)或SNARE蛋白SNAP25和Synaxin的相互作用而抑制。其次，将确定突触素家族在广泛的钙浓度范围内作为钙感受器的能力。利用生物化学和生物物理方法，对突触素I-XI膜和SNARE相互作用的钙依赖性进行了研究。最后，PC12细胞中主要的突触凝集素，其钙亲和力高于或低于突触凝集素I和IX，将在神经内分泌细胞中表达，以测试它们是否改变胞吐的钙敏感性。因此，我们将直接检验我们的假设，即给定细胞胞吐的钙敏感性反映了细胞内突触素亚型互补的钙亲和力。更好地了解突触聚集蛋白家族如何调节神经元胞吐作用将有助于突触传递的整体机制以及这一过程如何促进突触可塑性。对神经元胞吐调节的了解可能最终为突触传递受损的疾病的治疗提供靶点。

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