G-protein regulation of exocytotic transmitter release

描述(申请人提供)：通过调节胞吐作用释放化学递质是许多形式的细胞间通讯的基础，包括激素释放和突触传递。G蛋白偶联受体(GPCRs)协调胞吐作用的复杂调节，特别是抑制神经分泌细胞释放递质。抑制性GPCRs可以是自体或异质受体，通常是Gi/o偶联的，通过释放G蛋白β-伽马亚基(Gβ-伽马亚基)起作用。最广泛研究的抑制机制涉及电压门控钙通道(钙通道)的调节，但也有对胞吐机构的直接影响的报道。我们的初步数据显示，Gbeta-Gamma可以与SNAP25和Synaxin-1A结合，这表明Gbeta-Gamma可能同时针对钙通道和SNARs来抑制胞吐作用。这项提议的中心目标是剖析Gbeta-Gamma控制神经分泌细胞胞吐递质释放的分子基础。为了实现这一目标所需的精确生物物理分析，我们将使用肾上腺嗜铬细胞，这是一种提供显著实验优势的神经分泌模型。此外，嗜铬细胞释放的儿茶酚胺在应激或危险的协调反应中发挥着重要的生理作用。我们将结合碳纤维安培法、膜片钳电生理学和笼状化合物的闪光光解，以及新的分子工具(突变的Gβ-γ亚基和抑制肽)来剖析钙通道和SNARs在Gβ-γ介导的胞吐调节中的作用。在目标1中，我们将测试假设，即Gbeta-Gamma在钙通道和其他下游靶点平行作用，以抑制递质。在目标#2中，我们将描述Gbeta-Gamma与胞吐机制的相互作用，并验证Gbeta-Gamma通过与突触素-L竞争与SNAP25结合来抑制胞吐的假设。在目标#3中，我们将使用突变和肽图来表征Gbeta-Gamma-SNAP25的相互作用，并产生新的分子工具来剖析其在嗜铬细胞胞吐调节中的作用。综上所述，我们的研究将极大地提高对控制神经递质和激素分泌的分子机制的认识，并为与分泌和神经调节相关的疾病的病理基础提供洞察。

DESCRIPTION (provided by applicant): Release of chemical transmitters by regulated exocytosis underlies many forms of intercellular communication, including hormone release and synaptic transmission. G protein-coupled receptors (GPCRs) orchestrate complex regulation of exocytosis, and in particular inhibit transmitter release from neurosecretory cells. Inhibitory GPCRs can be auto- or hetero-receptors, are typically Gi/o coupled, and work by release of G protein beta-gamma subunits (Gbeta-gamma). The most intensively studied mechanism of inhibition involves modulation of voltage-gated Ca2+ channels (Ca-channels), but direct effects on the exocytotic apparatus have also been reported. Our preliminary data show that Gbeta-gamma binds to SNAP25 and syntaxin-1A, suggesting that Ca-channels and SNAREs might be targeted in parallel by Gbeta-gamma to inhibit exocytosis. The central goal of this proposal is to dissect the molecular basis by which Gbeta-gamma controls exocytotic transmitter release in neurosecretory cells. To enable the precise biophysical analyses required to address this goal, we will use adrenal chromaffin cells, a neurosecretory model that provides significant experimental advantages. Furthermore, catecholamines released from chromaffin cells play important physiological roles in the coordinated response to stress or danger. We will combine carbon fiber amperometry, patch clamp electrophysiology, and flash photolysis of caged compounds, along with novel molecular tools (mutant Gbeta-gamma subunits and inhibitory peptides) to dissect the roles of Ca-channels and SNAREs in Gbeta-gamma -mediated regulation of exocytosis. In aim #1 we will test the hypothesis that Gbeta-gamma acts in parallel at Ca-channels and other downstream targets to inhibit transmitter. In aim #2 we will characterize the interaction of Gbeta-gamma with the exocytotic machinery and test the hypothesis that Gbeta-gamma inhibits exocytosis by competing with synaptotagmin-l for binding to SNAP25. In aim #3 we will use mutational and peptide mapping to characterize the Gbeta-gamma - SNAP25 interaction and generate novel molecular tools to dissect its role in the regulation of chromaffin cell exocytosis. To summarize, our investigations will significantly advance knowledge of the molecular mechanisms that control neurotransmitter and hormone secretion, and provide insight into the pathological basis of diseases related to secretion and neuromodulation.