Defining the Role of Lipid Rafts in Neurotransmission

描述（由申请人提供）：SNARE蛋白形成调节胞吐作用所需的最小机制，这是神经系统内所有信息传递的基础过程。脂筏是一种动态的膜微结构域，作为调节膜功能的平台，与包括阿尔茨海默氏症和帕金森氏症在内的许多疾病有关。虽然已发现SNARE蛋白定位于脂筏，并且虽然脂筏的破坏导致受调节的胞吐的抑制，但脂筏在介导胞吐中发挥的确切作用尚未确定。该建议旨在：1）确定受调节的胞吐作用的具体阶段（例如，核心复合物形成、引发和融合），其中脂筏发挥重要作用; 2）确定脂筏在调节融合孔动力学中的功能;和3）确定参与神经元SNARE蛋白Syntaxin 1A靶向脂筏结构域的调节元件。该实验方法将利用完整和胆固醇耗尽的嗜铬细胞中的碳纤维安培法和膜电容测量的组合，以及异源表达各种野生型和突变SNARE蛋白的HEK细胞中的生化脂筏分离。

DESCRIPTION (provided by applicant): SNARE proteins form the minimal machinery required for regulated exocytosis, a process that underlies all information transfer within the nervous system. Lipid rafts, which have been implicated in a number of diseases including Alzheimer's and Parkinson's, are dynamic membrane microdomains that serve as platforms to regulate membrane function. While SNARE proteins have been found to localize to lipid rafts, and while disruption of lipid rafts results in the inhibition of regulated exocytosis, the exact role that lipid rafts exert in mediating exocytosis has not yet been determined. This proposal seeks to: 1) identify the specific stages of regulated exocytosis (e.g., core complex formation, priming, and fusion) in which lipid rafts play an important role; 2) determine the function of lipid rafts in modulating fusion pore dynamics; and 3) determine the regulatory elements involved in targeting of the neuronal SNARE protein Syntaxin 1A to lipid raft domains. The experimental approach will utilize a combination of carbon fiber amperometry and membrane capacitance measurements in both intact and cholesterol-depleted chromaffin cells, as well as biochemical lipid raft isolations in HEK cells heterologously expressing various wild-type and mutant SNARE proteins.