Regulation of tomosyn-SNARE complex assembly in neurosecretion

描述（由申请人提供）：SNARE蛋白组装成三元核心复合物对神经递质释放至关重要。SNARE复合物组装的精确调控最终决定了胞外事件的位置和动力学。我们的目标是了解调节这些SNARE复合物的时间和空间组装的机制。Tomosyn是一种蛋白，在确定浸润性SNARE复合物的水平中起关键作用。它的调节作用被认为主要是通过与Q-SNARE syntaxinl A的相互作用介导的，这种相互作用导致了非融合性SNARE复合物的形成。这项研究的目的是提供对神经递质释放调控中tomosyn-SNARE复合物组装/拆卸的分子机制和信号转导途径的理解。我们的一般假设是，Rho-GTPase信号通路促进了tomosyn-SNARE复合物的形成，并被蛋白激酶a依赖性通路拮抗，这些通路的激活平衡微调了融合能力强的SNARE复合物的水平。目的1将验证tomosyn-SNARE复合物的形成受到分泌激动剂的动态控制以调节分泌的假设。此外，我们将确定对这种调节重要的n端tomosyn结构域，以及内源性tomosyn对颗粒启动和胞外分泌的数量影响。Aim 2将验证一个假设，即tomosyn-SNARE复合物的水平及其对胞外通路的功能影响是由促进tomosyn-SNARE复合物组装的Rho激活状态和拮抗tomosyn-SNARE复合物组装的PKA激活状态之间的平衡所精细调节的。目的3将定义质膜上tomosyn-SNARE复合物的空间特性，并确定RhoA和PKA是否介导这些复合物的组装和拆卸的空间分隔效应。实验将使用生化、光学和电生理方法结合在肾上腺染色质细胞上进行，这是一种高度表征神经递质释放的细胞模型。了解神经递质释放的调节对于理解神经系统的功能至关重要，对于以特定神经递质失衡为典型的许多精神和神经疾病的治疗方法的发展至关重要。

DESCRIPTION (provided by applicant): The assembly of SNARE proteins into a ternary core complex is essential for neurotransmitter release. Precise regulation of SNARE complex assembly ultimately determines the site and dynamics of the exocytotic event. Our objective is to understand the mechanisms that regulate temporal and spatial assembly of these SNARE complexes. Tomosyn is a protein that is critical in setting the level effusion- competent SNARE complexes. Its regulatory action has been proposed to be primarily mediated by its interaction with the Q-SNARE syntaxinl A, which results in the formation of non-fusogenic SNARE complexes. The goal of the research proposed is to provide an understanding of the molecular mechanisms and signal transduction pathways governing the assembly/disassembly of tomosyn-SNARE complexes in the regulated release of neurotransmitter. Our general hypothesis is that tomosyn-SNARE complex formation is promoted by Rho-GTPase signaling pathways and antagonized by protein kinase A-dependent pathways, with the balance of activation of these pathways fine-tuning the level of fusion-competent SNARE complexes. Aim 1 will test the hypothesis that the formation of tomosyn-SNARE complexes is under dynamic control by secretory agonists to regulate secretion. In addition, we will identify N-terminal tomosyn domains important for this regulation, and quantity effects of endogenous tomosyn on granule priming and exocytosis. Aim 2 will test the hypothesis that the level of tomosyn-SNARE complexes and their functional effects on the exocytotic pathway are finely regulated by a balance between the activation state of Rho, to promote tomosyn-SNARE complex assembly, and PKA, to antagonize tomosyn-SNARE complex assembly. Aim 3 will define the spatial properties of tomosyn-SNARE complexes on the plasma membrane and determine if RhoA and PKA mediate spatially delimited effects on the assembly and disassembly of these complexes. Experiments will be performed using a combination of biochemical, optical and electrophysiological approaches on adrenal chromaffin cells, a highly characterized cell model for neurotransmitter release. Understanding the regulation of neurotransmitter release is essential to understanding the function of the nervous system and fundamental to development of therapeutic treatments in the many psychiatric and neurological conditions typified by an imbalance of particular neurotransmitters.