Role of CALHM1 ion channel in taste transduction

描述（由申请人提供）：我们提出的研究的总体目标是利用我们对CALHM1作为新型离子通道家族创始成员的身份的新见解。CALHM1是一种功能未知的基因，被确定为影响发病年龄的晚发性阿尔茨海默病的易感因素。CALHM1编码一种在整个大脑和味蕾中表达的膜蛋白，与其他功能特征蛋白缺乏显著的同源性，尽管已经鉴定出五种人类同源蛋白，并且CALHM1在物种间是保守的。我们最近发现CALHM1是Ca2+渗透离子通道的成孔亚基，具有不寻常的渗透特性和电压和细胞外Ca2+浓度（Ca2+o）的门控调节。我们发现CALHM1对于感知甜味、苦味和鲜味至关重要，因为敲除CALHM1的小鼠无法感知这些味道。此外，通过发现CALHM1是一个ATP渗透通道，我们已经确定了将CALHM1表达与味觉联系起来的分子机制，并且味觉剂通过CALHM1通道激活ATP释放作为一种神经递质，通过电压依赖机制将外周神经系统的味觉转导到中枢神经系统。我们将采用生物物理学（电生理学、光学成像）、生物化学和细胞生物学方法的结合来定义CALHM1在味觉中的分子生理学。我们将记录野生型小鼠和CALHM1基因缺失小鼠味觉细胞的电特性，并全面表征CALHM1通道在味觉细胞中的特性。我们将通过细胞内Ca2+的单细胞成像和全细胞电生理来确定CALHM1在味觉细胞信号转导中的作用。由于CALHM1通道是多聚体的，II型味觉细胞也表达CALHM2和CALHM3，并且CALHM1和calh3的共同表达产生了一个新的ATP可渗透通道，我们将确定所有三种CALHM1的生化和功能相互作用以及作用，以确定它们的通道特性，目的是重建味觉细胞中的ATP释放通道电流。最后，我们将使用异源细胞和味觉细胞的电生理记录来了解味觉细胞的电反应激活CALHM通道并被CALHM通道激活的机制。这些研究结果将为这种独特的电压门控Ca2+和ATP渗透离子通道的性质和调控及其在味觉感知中的重要作用提供新的见解。

DESCRIPTION (provided by applicant): The broad goal of our proposed studies is to exploit our new insights into the identity of CALHM1 as a founding member of a novel ion channel family. CALHM1, a gene of unknown function, was identified as a susceptibility factor for late-onset Alzheimer's disease that influences the age of onset. CALHM1 encodes a membrane protein expressed throughout the brain and in taste buds that lacks significant homology to other functionally characterized proteins, although five human homologs have been identified, and CALHM1 is conserved across species. We recently identified CALHM1 as the pore-forming subunit of Ca2+ permeable ion channel with unusual permeation properties and gating regulation by both voltage and extracellular Ca2+ concentration (Ca2+o). We have discovered that CALHM1 is essential for the perceptions of sweet, bitter and umami tastes, since CALHM1 knockout mice cannot perceive these tastants. Furthermore, we have identified the molecular mechanism that links CALHM1 expression to taste perception by discovering that CALHM1 is an ATP permeable channel, and that tastants activate ATP release as a neurotransmitter through CALHM1 channels by a voltage dependent mechanism that transduces taste in the periphery to the central nervous system. We will employ a combination of biophysical (electrophysiology, optical imaging), biochemical and cell biological approaches to define the molecular physiology of CALHM1 in taste perception. We will record the electrical properties of taste cells from wild-type mice and mice with CALHM1 genetically deleted and fully characterize the properties of CALHM channels in taste cells. We will define the role of CALHM1 in taste cell signal transduction by single cell imaging of intracellular Ca2+ and whole cell electrophysiology. Because a CALHM1 channel is multimeric, type II taste cells also express CALHM2 and CALHM3, and co-expression of CALHMs 1 and 3 generates a novel ATP-permeable channel, we will determine the biochemical and functional interactions and roles of all three CALHMs to define their channel properties with the goal to reconstitute the ATP release channel currents in taste cells. Finally we will use electrophysiological recordings of heterologous cells and taste cells to understand the mechanisms by which taste cell electrical responses activate CALHM channels and are in turn modified by CALHM channel activation. The results of these studies will provide new insights into the properties and regulation of this unique voltage-gated Ca2+ and ATP permeable ion channel and its essential role in taste sensory perception.