G-protein regulation of exocytotic transmitter release

描述（由申请人提供）：通过调节胞外分泌释放化学递质是多种细胞间通讯的基础，包括激素释放和突触传递。G蛋白偶联受体（gpcr）协调胞外分泌的复杂调控，特别是抑制神经分泌细胞的递质释放。抑制性gpcr可以是自受体或异受体，通常是Gi/o偶联的，通过释放G蛋白β - γ亚基（gβ - γ）起作用。最深入研究的抑制机制涉及调节电压门控Ca2+通道（ca通道），但也有报道直接影响胞外装置。我们的初步数据显示，gβ - γ与SNAP25和syntaxin-1A结合，表明gβ - γ可能同时靶向ca通道和SNAREs以抑制胞吐作用。本提案的中心目标是剖析gβ - γ控制神经分泌细胞的胞外递质释放的分子基础。为了实现实现这一目标所需的精确生物物理分析，我们将使用肾上腺染色质细胞，这是一种具有显著实验优势的神经分泌模型。此外，从染色质细胞释放的儿茶酚胺在对压力或危险的协调反应中发挥重要的生理作用。我们将结合碳纤维电流法、膜片钳电生理学和笼中化合物的闪光光解，以及新的分子工具（突变gβ - γ亚基和抑制肽）来剖析ca通道和SNAREs在gβ - γ介导的胞外分泌调节中的作用。在目标#1中，我们将测试gβ - γ平行作用于ca通道和其他下游目标以抑制发射器的假设。在目标#2中，我们将描述gβ - γ与胞吐机制的相互作用，并验证gβ - γ通过与synaptotagmin- 1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作图来表征gβ - γ - SNAP25相互作用，并生成新的分子工具来剖析其在染色质细胞胞外分泌调节中的作用。总之，我们的研究将显著促进对控制神经递质和激素分泌的分子机制的认识，并为了解与分泌和神经调节有关的疾病的病理基础提供见解。

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.