Role of CALHM1 ion channel in taste transduction

描述(由申请人提供)：我们提议的研究的广泛目标是利用我们对CALHM1作为一个新的离子通道家族的创始成员的身份的新见解。CALHM1是一个功能未知的基因，被认为是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，该蛋白与其他功能特征的蛋白缺乏显著的同源性，尽管已经确定了五个人类同源物，并且CALHM1在物种之间是保守的。我们最近发现CALHM1是钙离子通透性离子通道的致孔亚单位，具有特殊的通透性和受电压和细胞外钙离子浓度(Ca2+o)的门控调节。我们已经发现，CALHM1对于感知甜、苦和鲜味是必不可少的，因为CALHM1基因敲除的小鼠无法感知这些味道。此外，我们还发现CALHM1是一个ATP通透通道，品尝者通过电压依赖的机制通过CALHM1通道激活ATP释放作为神经递质，将外周的味觉传递到中枢神经系统，从而确定了CALHM1表达与味觉之间的联系的分子机制。我们将结合生物物理学(电生理学、光学成像)、生物化学和细胞生物学的方法来定义CALHM1在味觉感知中的分子生理学。我们将记录野生型小鼠和CALHM1基因缺失小鼠的味觉细胞的电特性，并全面表征味觉细胞中CALHM通道的特性。我们将通过对细胞内钙离子的单细胞成像和全细胞电生理学来确定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CALHM3的共同表达产生了一种新的ATP通透通道，因此我们将确定这三种CALHM的生化和功能相互作用及其作用，以确定它们的通道特性，目标是重建味觉细胞中的ATP释放通道电流。最后，我们将利用异种细胞和味觉细胞的电生理记录来了解味觉细胞电反应激活CALHM通道并进而被CALHM通道激活所改变的机制。这些研究结果将为这一独特的电压门控钙和三磷酸腺苷通透离子通道的性质和调节及其在味觉知觉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.