Molecular physiology of CALHM ion channels

我们提出的研究的广泛目标是利用我们对分子机制的新见解， CALHM 1和CALHM 3作为味觉感知中的新型离子通道的组分的生理作用。我们 发现CALHM 1是一种膜蛋白，在整个大脑和味蕾中表达， 与其他蛋白质具有显著的同源性，尽管已经鉴定了五种同源物，并且CALHM 1是 在物种间是保守的。我们鉴定了CALHM 1作为一个离子通道的成孔亚基， 孔径和电压和细胞外Ca 2+（Ca 2 +o）的门控调节。我们发现CALHM 1是 由于CALHM 1基因敲除，II型味蕾细胞对甜味、苦味和鲜味的感知至关重要。 小鼠不能感知这些促味剂。我们通过发现CALHM 1是一种 电压门控的ATP-渗透性离子通道，以及促味剂诱发的Na+动作电位触发ATP释放 作为一种神经递质，通过CALHM 1相关的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，CALHM 3是天然的 电压门控ATP释放通道，作为一个孔形成亚基与CALHM 1，以创建一个 II型细胞中的异聚ATP释放通道。CALHM 3的遗传缺失也消除了 小鼠感知甜、苦和鲜味物质。离子的分子机制和结构基础 尽管CALHM通道的渗透和门控具有生理学重要性，但尚未了解它们。也不是 了解CALHM 3整合到CALHM 1/3通道中如何强烈影响电压门控激活， 这是一个允许CALHM 1/3通道响应动作电位的关键特征。温度显著影响 味觉与生理和享乐主义的影响，但周边的味道转导机制的贡献，温度的感知和感觉的味道的影响，主要是 未知我们已经发现，温度显著影响CALHM 1/3电导以及 II型细胞的电兴奋性。我们将在天然味蕾细胞中采用电生理学， 异源表达系统，诱变，冷冻EM，和建模，以确定门控机制， CALHM 1和CALHM 1/3通道，CALHM 3作为孔形成亚基如何增强电压门控性 CALHM 1/3通道的激活，以及CALHM 1/3通道如何响应由 在很宽的温度范围内都能产生味觉刺激。使用一种新的小鼠模型，其中CALHM 1/3在 味蕾细胞已经被设计成具有不同的温度敏感性，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参与了温度的影响 对味觉敏感度和感知的影响。

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.