Molecular physiology of CALHM ion channels

我们提议的研究的广泛目标是利用我们对分子机制和 CALHM1和CALHM3作为一种新的离子通道成分在味觉中的生理作用。我们 发现CALHM1是一种膜蛋白，它在整个大脑和缺乏 与其他蛋白质有显著的同源性，尽管已经鉴定出五个同源物，CALHM1是 在不同物种之间保存。我们确定CALHM1是离子通道的一个成孔亚单位，具有一个大的 孔径和电压及细胞外钙离子(钙离子)对门控的调节。我们发现CALHM1是 自CALHM1基因敲除以来，II型味蕾细胞对感知甜味、苦味和鲜味至关重要 老鼠察觉不到这些味道。我们通过发现CALHM1是一种 电压门控的ATP通透离子通道和味觉诱发的Na+动作电位触发ATP释放 作为一种神经递质，通过CALHM1相关通道从外周传递味觉信息 到中枢神经系统。我们进一步发现，CALHM3是天然的 电压门控的ATP释放通道，作为与CALHM1一起形成孔的亚单位，创建 II型细胞中的异构体ATP释放通道。CALHM3的基因缺失也消除了 老鼠能感知甜味、苦味和鲜味物质。离子的分子机理和结构基础 尽管CALHM通道具有重要的生理意义，但其渗透和门控机制尚不清楚。也不是 了解将CALHM3集成到CALHM1/3通道如何如此强烈地影响电压门控激活， 一种允许CALHM1/3通道对动作电位作出反应的关键特性。温度显著影响 味觉具有生理和享乐主义的含义，但外周味觉转导机制对温度对味觉和味觉的影响的贡献很大 未知。我们发现，温度对CALHM1/3电导和CALHM1/3电导有显著影响。 II型细胞的电兴奋性。我们将在天然味蕾细胞中应用电生理学 异源表达系统，突变，冷冻-EM和建模，以定义门控机制 CALHM1和CALHM1/3通道，CALHM3作为成孔亚基如何增强电压门控 CALHM1/3通道的激活以及CALHM1/3通道如何对 在广泛的温度范围内进行味觉刺激。使用一种新的小鼠模型，在该模型中CALHM1/3在 味蕾细胞已经被设计成具有明显的温度敏感性，我们将定义如何区分 温度对ATP释放通道门控和兴奋性的影响可能提供了一种机制 外周味觉系统中的温度敏感通道有助于温度的影响 在味觉敏感度和知觉上。

The broad goal of our proposed studies is to exploit our new insights into the molecular mechanisms and physiological roles of CALHM1 and CALHM3 as components of a novel ion channel in taste perception. We discovered CALHM1 as a membrane protein that expressed throughout the brain and in taste buds that lacks significant homology to other proteins, although five homologs have been identified, and CALHM1 is conserved across species. We identified CALHM1 as a pore-forming subunit of an ion channel with a large pore diameter and gating regulation by voltage and extracellular Ca2+ (Ca2+o). We discovered that CALHM1 is essential for perceptions of sweet, bitter and umami tastes by type II taste bud cells, since CALHM1-knockout mice cannot perceive these tastants. We identified the essential role of CALHM1 by discovering that it is a voltage-gated ATP-permeable ion channel, and that tastant-evoked Na+ action potentials trigger ATP release as a neurotransmitter through CALHM1-associated channels to transduce taste information from the periphery to the central nervous system. We further discovered that CALHM3 is an essential component of the native voltage-gated ATP-release channel, contributing as a pore-forming subunit with CALHM1 to create a heteromeric ATP-release channel in type II cells. Genetic deletion of CALHM3 also eliminates the ability of mice to perceive sweet, bitter and umami substances. The molecular mechanisms and structural bases of ion permeation and gating of CALHM channels are not understood despite their physiological importance. Nor is it understood how integration of CALHM3 into a CALHM1/3 channel so strongly affects voltage-gated activation, a key feature that allows CALHM1/3 channels to respond to action potentials. Temperature notably influences taste perception with physiological and hedonistic implications, but the contribution of peripheral taste-transduction mechanisms to the effects of temperature on the perception and sensation of tastes is largely unknown. We have discovered that temperature strikingly influences CALHM1/3 conductance as well as the electrical excitability of type II cells. We will employ electrophysiology in native taste bud cells and heterologous expression systems, mutagenesis, cryo-EM, and modeling to define the gating mechanisms of CALHM1 and CALHM1/3 channels, how CALHM3 as a pore-forming subunit enhances voltage-gated activation of CALHM1/3 channels, and how CALHM1/3 channels respond to action potentials evoked by tastant stimulation over a wide range of temperatures. Using a novel mouse model in which CALHM1/3 in taste bud cells has been engineered to have distinct temperature sensitivity, we will define how differential effects of temperature on ATP-release channel gating and excitability may provide a mechanism for how a temperature-sensitive channel in the peripheral gustatory system contributes to the influence of temperature on taste sensitivity and perception.