TRPV4: Mechanosensitivity and Skeletal Dysplasia

描述(申请人提供)：与基于GPCR的视觉、嗅觉和味觉不同，听觉、触觉和血压或全身渗透压感知的分子机制尚不清楚。除了细菌通道MSCL、MSCS外，离子通道是如何接受机械力的仍不清楚。我们正在使用RAT TRP通道V4亚型，以帮助填补这一知识空白。出乎意料的是，我们现在展示了在非洲爪哇卵母细胞膜片钳下，TRPV4的S对膜拉伸的直接和即时的反应。我们还开发了酵母作为一个新的领域来剖析TRPV4。我们的工作挑战了占主导地位的TRPV4‘S渗透门控模型。我们计划使用战略性的多肽插入、截断和嵌合体来定义TRPV4中的力感知域。我们还将通过检查膜插层化合物和突变假定的脂类锚定氨基酸的效果来测试来自脂类的力理论。2008-10发现TRPV4功能获得(GOF)突变可导致人类骨骼发育病理(短小畸形症、化生性发育不良等)。为了鉴定和理解这些疾病等位基因造成的分子缺陷，我们将阐明TRPV4的S额外的门控机制，并将利用改进的电生理技术和基于酵母的表型分析方法详细地检测所有7个人类GOF等位基因。

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.