TRPV4: Mechanosensitivity and Skeletal Dysplasia

描述（由申请人提供）：与基于gpcr的视觉、嗅觉和味觉不同，听觉、触觉、血压或全身渗透压传感的分子机制尚不清楚。除了细菌通道外，离子通道如何接受机械力仍然不清楚。我们正在使用大鼠TRP通道V4亚型来帮助填补这一知识空白。出乎意料的是，我们现在已经证明了TRPV4在爪蟾卵母细胞膜片钳下对膜拉伸的直接和即时反应。我们还开发了酵母作为解剖TRPV4的新场所。我们的工作是对占主导地位的TRPV4渗透门控模型的挑战。我们计划使用战略性肽插入、截断和嵌合来定义TRPV4中的力感应结构域。我们还将通过检查膜插入化合物的作用和通过突变假定的脂质锚定氨基酸来测试脂质力理论。2008-10发现TRPV4功能获得（GOF）突变可导致人类骨骼发育病理（短肌缺损、萎缩性发育不良等）。为了描述和理解由这些疾病等位基因引起的分子缺陷，我们将阐明TRPV4的附加门控机制，并将利用改进的电生理技术和基于酵母的表型分析对所有7个人类GOF等位基因进行分子细节研究。

DESCRIPTION (provided by applicant): Unlike the GPCR-based vision, smell and tastes, the molecular mechanisms of hearing, touch, and blood-pressure or systemic-osmolarity sensing are unknown. How ion channels receive mechanical forces remain obscure, except for bacterial channels MscL, MscS. We are using the rat TRP channel V4 subtype, to help fill this knowledge gap. Against expectation, we have now shown TRPV4's direct and instant response to membrane stretch under Xenopus oocyte patch clamp. We have also developed yeast as a new arena to dissect TRPV4. Our work is challenging the dominant TRPV4's osmotic- gating model. We plan to define the force-sensing domain within TRPV4, using strategic peptide insertions, truncations and chimeras. We will also test the force-from-lipid theory by examining the effects of membrane-intercalating compounds and by mutating putative lipid-anchoring amino acids. 2008-10 found TRPV4 gain-of-function (GOF) mutations to cause human bone-development pathologies (brachyolmia, metatropic dysplasia, etc.). To characterize and understand the molecular defects caused by these disease alleles, we will elucidate TRPV4's additional gating mechanisms, and will examine all seven human GOF alleles in molecular detail with improved electrophysiological techniques and yeast-based phenotyping.