Cellular Physiology of Otopetrin Proton Channels

项目摘要/摘要 拟议的实验旨在阐明新发现的离子通道家族Otopetrins(OTOPS)的结构和功能特性。Otop是真核细胞中描述的仅次于Hv1的第二类质子选择性离子通道。Otop1最早是在前庭系统中发现的，在前庭系统中，它是发展碳酸钙耳锥所必需的，这种耳锥使毛细胞能够检测重力和加速度的变化。随后，基于对味觉细胞中丰富的检测酸味的转录本的功能分析，它在质子通道的无偏筛选中被鉴定出来。当Otop1在非洲爪哇卵母细胞和HEK-293细胞中表达时，它会产生一种质子选择性离子电流，该电流被细胞外的锌[2]阻断。哺乳动物基因组编码Otop1的两个同源物(Otop2和Otop3)，这两个同源物也形成具有不同功能特性的质子渗透离子通道。在这里，我们提出了一系列实验来定义OTOP通道的门控和渗透机制，并确定潜在的结构元素。Otop1和Otop3的低温EM结构使我们确定了几种可能的质子渗透途径；特定残基在渗透或锌[2]抑制中的作用将通过定点突变和膜片钳电生理学进行询问。其他实验将解决OTOP通道是否门控这一悬而未决的问题，并确定门控的结构决定因素。Otopetrin基因家族的成员广泛表达于全身，包括味觉、前庭和免疫系统、胃肠道和棕色脂肪组织。通过了解每种OTOP亚型的结构和功能特性，我们将能够更好地了解它们对细胞生理学的贡献。此外，离子通道是药物的首选靶标，而破坏其功能的突变是越来越多的遗传性或获得性疾病(通道病)的基础。

PROJECT SUMMARY/ABSTRACT The proposed experiments are aimed at elucidating the structural and functional properties of a newly discovered family of ion channels, the Otopetrins (Otops). The Otops are just the second class, after Hv1, of proton-selective ion channels described in eukaryotic cells. Otop1 was first discovered in the vestibular system where it is required for the development of calcium carbonate-based otoconia that allow hair cells to detect changes in gravitational forces and acceleration. It was subsequently identified in an unbiased screen for proton channels based on functional analysis of transcripts enriched in taste cells that detect sour. When expressed in Xenopus oocytes and HEK- 293 cells, Otop1 generates a proton-selective ion current that is blocked by extracellular Zn[2+]. The mammalian genome encodes two homologs of Otop1 (Otop2 and Otop3), which also form proton permeable ion channels with distinct functional properties. Here we propose a series of experiments to define mechanisms of gating and permeation of Otop channels, and to identify the underlying structural elements. The CryoEm structure of Otop1 and Otop3 have led us to identify several possible proton permeation pathways; the function of specific residues in permeation or Zn[2+] inhibition will be interrogated using site directed mutagenesis and patch clamp electrophysiology. Other experiments will address the outstanding question of whether Otop channels are gated and identify structural determinants for gating. Members of the otopetrin gene family are widely expressed throughout the body, including in the taste, vestibular and immune systems, in the gastrointestinal tract, and in brown adipose tissue. By understanding the structural and functional properties of each of the Otop isoforms, we will be better able to understand how they contributes to cellular physiology. Moreover, ion channels are the preferred targets for pharmaceuticals and mutations that disrupt their function underlie a growing number of inherited or acquired disorders (channelopathies).