Intramolecular interactions and regulation of TRP channels

哺乳动物瞬时受体电位（TRP）超家族由28个阳离子通道组成，分为6个亚家族，通过对光、力、温度和化学物质等多种刺激的反应参与感觉生理学。TRP蛋白，虽然具有适度的序列同源性，但共享重要的结构组织，例如具有六个跨膜结构域和四聚体组装，主要使用冷冻电子显微镜显示。然而，人们对配体、药物和突变（作为“输入”）如何诱导TRP分子内相互作用和蛋白质构象的变化知之甚少，它们通过这些变化调节TRP通道功能（作为“输出”），这阻碍了治疗干预。 在过去的几年里，我的研究生/本科生和博士后研究员在理解几个TRP成员的功能和调节方面取得了重大进展，主要来自TRP多囊蛋白（TRPP）和香草素（TRPV）亚家族。他们的初步实验揭示了TRPP 3和TRPV 6中新的和功能上重要的分子内相互作用，这对于制定我们的假设和启动该计划至关重要。 该计划的总体目标是研究TRPP 3和TRPV 6中的分子内相互作用以及构象变化如何通过配体和突变介导通道功能的调节，通过这些来训练HQP。为此，我们将在三个项目中进行实验：1）确定TRPP 3细胞内连接体及其磷酸化在调节通道功能和介导磷脂作用中的作用，2）表征TRPV 6成孔膜螺旋之间的相互作用及其如何介导磷脂的功能刺激，和3）检查TRPP 3成孔螺旋之间的相互作用，其介导磷脂的功能抑制。方法论将使用青蛙卵母细胞和哺乳动物细胞中的电生理学来评估通道功能。通道蛋白内的相互作用和构象变化将通过蛋白质-蛋白质相互作用、免疫荧光和计算机模拟进行研究（与J. B.彭，亚拉巴马大学伯明翰分校），等等。TRPP 3通道的结构-功能关系将通过电生理学和冷冻电子显微镜（与Dr E. Cao，Univ of犹他州）。 意义通过使用最先进的跨学科方法，该计划将获得对TRPP 3和TRPV 6分子内相互作用和构象变化如何通过配体和突变介导通道功能调节的共享或不同机制的新见解。从这项研究中获得的知识也将有助于了解其他TRP通道，并可能为未来的治疗干预提供新的靶点。该计划将由HQP学员通过当地和国家/国际合作进行，从而为他们提供动态和富有成效的培训环境。

Mammalian transient receptor potential (TRP) superfamily has 28 cation channels that are grouped into six subfamilies and implicated in sensory physiology by responding to various stimuli including light, force, temperature and chemicals. TRP proteins, though with modest sequence homology, share significant structural organizations, eg with six transmembrane domains and tetrameric assembly, mostly revealed using cryo-electron microscopy. However, little is known about how ligands, drugs and mutations (as `inputs') induce changes in TRP intramolecular interactions and protein conformation through which they regulate TRP channel function (as `output'), which hampers therapeutic interventions. For the past years my graduate/undergraduate students and postdoct fellows have made significant progress on understanding the function and regulation of several TRP members, mainly from the TRP polycystin (TRPP) and vanilloid (TRPV) subfamilies. Their preliminary experiments revealed novel and functionally important intramolecular interactions in TRPP3 and TRPV6, which is critical for formulating our hypotheses and initiating this program. The overall OBJECTIVE of this program is to study how intramolecular interactions in TRPP3 and TRPV6 and conformational changes mediate regulation of channel function by ligands and mutations, through which HQP will be trained. For this goal we will carry out experiments in three projects: 1) determining roles of TRPP3 intracellular Linker and its phosphorylation in regulating channel function and in mediating the effect of phospholipid, 2) characterizing interaction between TRPV6 pore-forming membrane helices and how it can mediate functional stimulation by phospholipid, and 3) examining interaction between TRPP3 pore-forming helices, which mediates functional inhibition by phospholipid. METHODOLOGY. Channel function will be assessed using electrophysiology in frog oocytes and mammalian cells. Interactions and conformational changes within a channel protein will be examined by protein-protein interaction, immunofluorescence and computer simulation (in collaboration with Dr J.B. Peng, Univ of Alabama at Birmingham), among others. Structure-function relationships of TRPP3 channel will be examined by electrophysiology and cryo-electron microscopy (in collaboration with Dr E. Cao, Univ of Utah). SIGNIFICANCE. By using state-of-the-art interdisciplinary approaches this program will gain novel insights into shared or distinct mechanisms underlying how TRPP3 and TRPV6 intramolecular interactions and conformational changes mediate regulation of channel function by ligands and mutations. Knowledge gained from this study will also help understanding other TRP channels and may provide novel targets for future therapeutic interventions. This program will be carried out by HQP trainees via local and national/international collaborations and will thus provide dynamic and productive training environments for them.