Molecular physiology of CALHM ion channels

CALHM 离子通道的分子生理学

• 批准号: 10192500
• 负责人: James Kevin FOSKETT
• 金额: $ 52.56万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10192500
• 关键词: Action Potentials; Address; Affect; Brain; Caliber; Charge; Cryoelectron Microscopy; Dependence; Diabetes Mellitus; Electrophysiology (science); Elements; Engineering; Esthesia; Evoked Potentials; Family; Food; Formulation; G-Protein-Coupled Receptors; Genetic; Goals; Human; Ion Channel; Ions; Kinetics; Knockout Mice; Life; Mediating; Medicine; Membrane Proteins; Methods; Modeling; Molecular; Mus; Mutagenesis; Nerve; Neuraxis; Neurons; Neurotransmitters; Nutritional; Obesity; Pathway interactions; Perception; Peripheral; Permeability; Pharmacology; Physiological; Physiology; Play; Proteins; Regulation; Role; Scheme; Signal Transduction; Structure; System; TRPM5 gene; Taste Bud Cell; Taste Buds; Taste Perception; Temperature; Type II Epithelial Receptor Cell; base; extracellular; insight; mouse model; mutant; neurotransmitter release; novel; response; taste system; taste transduction; voltage

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.

我们提出的研究的广泛目标是利用我们对分子机制和的新见解 CalHM1和CalHM3的生理作用是味觉感知中新型离子通道的组成部分。我们 发现Calhm1是一种在整个大脑中表达的膜蛋白，在缺乏味蕾中 尽管已经确定了五个同源物，但与其他蛋白质的重要​​同源性，Calhm1是 跨物种保守。我们将Calhm1识别为一个离子通道的孔单元， 通过电压和细胞外Ca2+（Ca2+ O）的孔径和门控调节。我们发现Calhm1是 对于II型味蕾细胞对甜，苦和鲜味的口味的感知至关重要，因为CALHM1-KNOCKOUT 小鼠无法感知这些味道。我们通过发现它是一个 电压门控ATP可渗透的离子通道，并诱发的Na+动作电位触发ATP释放 作为神经递质通过CALHM1相关的通道，从外围传递口味信息 到中枢神经系统。我们进一步发现calhm3是本地的重要组成部分 电压门控ATP释放通道，用Calhm1成为孔形成子单位以创建一个 II型细胞中的杂体ATP释放通道。 CALHM3的遗传缺失也消除了 小鼠感知甜，苦和鲜味的物质。离子的分子机理和结构碱基 Calhm通道的渗透和门控具有生理意义。也不是 了解将CalHM3整合到CALHM1/3通道中如何强烈影响电压门控激活， 允许CALHM1/3通道可以响应动作电位的关键功能。温度明显影响 具有生理和享乐主义意义的味觉感知，但是外周味道转化机制对温度对感知和感觉的影响的贡献在很大程度上是 未知。我们发现温度显着影响CALHM1/3电导以及 II型细胞的电兴奋性。我们将在天然味蕾细胞中使用电生理学，并 异源表达系统，诱变，冷冻EM和建模，以定义门控机制 calhm1和calhm1/3通道，calhm3作为形成孔的亚基如何增强电压门控 CALHM1/3通道的激活，以及CALHM1/3通道如何响应动作电位。 在广泛的温度下刺激味道。使用新型鼠标模型，其中calhm1/3在 味蕾细胞已经设计为具有独特的温度灵敏度，我们将定义差异 温度对ATP释放通道门控和兴奋性的影响可能为如何提供一种机制 周围味道系统中温度敏感的通道有助于温度的影响 在口味敏感性和感知上。