Identification of CALHM proteins as ion channels

项目总结 我们提议的研究的目标是确定符合IDG条件的CALHM4、CALHM5和CALHM6是否 离子通道或离子通道亚单位。CALHM(前身为FAM26)基因家族由六个同源基因组成。 我们先前发现了CALHM1和CALHM3 AS的分子机制和生理作用 新的离子通道。CALHM1编码一种在整个大脑和味蕾中表达的膜蛋白 这与其他蛋白质缺乏显著的同源性，尽管已鉴定出5个同源性为20%-44% 序列相似性，CALHM蛋白在物种之间是保守的。我们确认CALHM1是一个毛孔- 大孔径离子通道亚基的形成及电压门控和胞外调节 Ca~(2+)(Ca~(2+))。我们发现，CALHM1对于感知甜味、苦味和鲜味是必不可少的 第二类味蕾细胞，因为CALHM1基因敲除的小鼠无法感知这些味觉。我们确定了 CALHM1通过发现它是一种电压门控的ATP通透离子通道而发挥重要作用，并且 味觉诱发的Na+动作电位通过CALHM1相关的神经递质激活ATP的释放 将味觉信息从外周传递到中枢神经系统的通道。我们进一步 研究发现，虽然CALHM3的表达不会产生新的离子通道，但它是一种必不可少的 天然电压门控三磷酸腺苷释放通道的组成部分，作为成孔亚单位与 CALHM1在II型细胞中创建异六聚体的ATP释放通道。CALHM3的基因缺失也 消除小鼠感知甜味、苦味和鲜味物质的能力。这些结果表明，其他 CALHM同系物也是离子通道或离子通道亚基，但没有关于它们的信息 分子功能。我们假设CALHM4、CALHM5和CALHM6是离子通道，而且 CALHM家族代表了参与嘌呤能信号传递的一系列ATP释放通道 遍及全身。我们将利用我们的洞察力和技术方法来阐明 用于确定CALHM4、CALHM5和CALHM6是离子通道还是离子的CALHM1和CALHM3 通道子单元。我们将使用电生理学、光学成像、三磷酸腺苷释放分析和生化 检验这一重要假说的方法。我们研究的影响预计将是发现 CALHM4、CALHM5和CALHM6的功能。在确定CALHM1和CALHM3为离子时， 我们的发现有望提供CALHM是否是一个离子家族的信息 通道蛋白。新离子通道的发现有望带来对细胞和 组织生理学，就像我们之前在味蕾和味觉方面所做的工作一样，随着 CALHM1和CALHM3的功能。因此，鉴定其他CALHM同系物的角色可以 提供新的生理学见解和治疗机会。

PROJECT SUMMARY The goal of our proposed studies is to determine whether IDG-eligible CALHM4, CALHM5 and CALHM6 are ion channels or ion channel subunits. The CALHM (formerly FAM26) gene family is comprised of six homologs. We previously discovered the molecular mechanisms and physiological roles of CALHM1 and CALHM3 as novel ion channels. CALHM1 encodes a membrane protein expressed throughout the brain and in taste buds that lacks significant homology to other proteins, although five homologs have been identified with 20-44% sequence similarity, and CALHM proteins are 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 whereas CALHM3 expression does not confer a novel ion channel, it is an essential component of the native voltage-gated ATP-release channel, contributing as a pore-forming subunit with CALHM1 to create a hetero-hexameric ATP-release channel in type II cells. Genetic deletion of CALHM3 also eliminates the ability of mice to perceive sweet, bitter and umami substances. These results suggest that other CALHM homologs are also ion channels or ion channel subunits, but there is no information regarding their molecular function. We hypothesize that CALHM4, CALHM5 and CALHM6 are ion channels, and furthermore that the CALHM family represents a family of ATP-release channels the contributes to purinergic signaling throughout the body. We will exploit our insights and technical approaches used to elucidate functions of CALHM1 and CALHM3 to determine whether CALHM4, CALHM5 and CALHM6 are ion channels or ion channel subunits. We will employ electrophysiology, optical imaging, ATP-release assays and biochemical approaches to test this overarching hypothesis. The impact of our research is expected to be the discovery of the functions of CALHM4, CALHM5 and CALHM6. Given the identification of CALHM1 and CALHM3 as ion- channel subunits, our findings are expected to provide information about whether CALHMs are a family of ion channel proteins. Identification of novel ion channels is expected to lead to new understanding of cell and tissue physiology, as our previous work in taste buds and taste perception did with the discoveries of the functions of CALHM1 and CALHM3. Accordingly, identification of the roles of the other CALHM homologs may inform new physiological insights and therapeutic opportunities.