Structure and function of Transient Receptor Potential Channels

瞬时感受器电位通道的结构和功能

基本信息

批准号：
10583880
负责人：
Alexander Sobolevsky
金额：
$ 46.05万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2028-02-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10583880
关键词：
Affinity Algae Architecture Binding Biochemical Biological Assay Biophysics Borates Calcium Calmodulin Cell Line Cells Chemicals Cloning Complex Cryoelectron Microscopy Data Disease Drug Design Drug Targeting Electrophysiology (science)Elements Epithelium Eukaryotic Cell Evolution Family Fluorescence Funding Genetic Polymorphism Genetic Variation Goals Haplotypes Hearing High temperature of physical object Human Image Inhibition of Apoptosis Inhibition of Cell Proliferation Integral Membrane Protein Ions Knowledge Length Lipids Malignant Neoplasms Mechanical Stress Mediating Membrane Methods Modernization Molecular Molecular Conformation Molecular Sieve Chromatography Mutagenesis Mutation Nociception Pain Pathogenesis Pattern Perception Permeability Phosphatidylinositol 4,5-Diphosphate Physiological Piperazines Predisposition Prognostic Marker Protein Engineering Rattus Regulation Role Ruthenium Red Sensory Site Smell Perception Specificity Spectrum Analysis Squirrel Stimulus Structure System TRP channel TRPV1 gene Taste Perception Techniques Temperature Therapeutic Tissues Touch sensation Vanilloid Variant Vision Visualization Work X-Ray Crystallography base cancer therapy chemical synthesis experience gain of function genetic variant human disease human genome sequencing hypercalciuria improved in silico inhibitor mimetics molecular dynamics mutant new therapeutic target novel therapeutic intervention novel therapeutics overexpression renal calcium sensor small molecule success uptake voltage

项目摘要

PROJECT SUMMARY Transient Receptor Potential (TRP) channels represent polymodal cellular sensors, which integrate chemical, temperature, mechanical stress and membrane voltage stimuli and convert them into ionic currents to regulate our senses of vision, hearing, taste, smell and touch and contribute to the perception of temperature and pain. TRP channels are implicated in the pathogenesis of numerous human diseases, including cancers, and represent one of the most ardently pursued drug targets. Despite recent successes in TRP channel structure determination, understanding of their genetic diversity, function and regulation is still far from being complete. Such limited knowledge represents a critical barrier to devising therapeutic strategies based on TRP channel regulation and to the progress in the rational drug design. We plan to study TRP channel structure and function using a combination of different biophysical and biochemical methods. Our specific aims are: 1) establish molecular bases of TRPV6 polymorphisms and disease variants, 2) determine structural mechanisms of TRPV6 inhibition, and 3) identify structural elements underlying similarities and difference in gating and regulation of TRPV6 and other TRP channels. TRP channels are challenging targets for structure-functional studies because they represent multimeric integral membrane proteins of a large size with typically low expression levels. To achieve our goals, we will use a combination of structural and functional approaches, including modern cryo- electron microscopy (cryo-EM), X-ray crystallography, protein engineering, Fluorescence-based Size Exclusion Chromatography (FSEC), calcium imaging, fluorescent spectroscopy and electrophysiology. We will express TRP channels, their mutants and genetic variants in eukaryotic cell lines, purify them using different membrane mimetic systems, and determine cryo-EM and crystal structures in the presence of different stimuli. We will then combine the nascent structural information with functional data to discern molecular mechanisms of TRP channel gating, inhibition and regulation by Ca2+, temperature and lipids. Achieving our aims will significantly improve understanding of TRP channel structure and function, resulting in a new dynamic template for theoretical prediction, in silico fitting and chemical synthesis of new drugs.

项目概要瞬时受体电位 (TRP) 通道代表多模式细胞传感器，它集成了化学、温度、机械应力和膜电压刺激，并将它们转换成离子电流来调节我们的视觉、听觉、味觉、嗅觉和触觉感知温度和疼痛。 TRP 通道与多种人类疾病（包括癌症）的发病机制有关代表最热烈追求的药物靶点之一。尽管最近 TRP 渠道结构取得了成功对它们的遗传多样性、功能和调控的确定、了解还远未完成。这种有限的知识是设计基于 TRP 通道的治疗策略的关键障碍监管和合理药物设计的进展。我们计划研究TRP通道的结构和功能结合使用不同的生物物理和生化方法。我们的具体目标是：1）建立 TRPV6多态性和疾病变异的分子基础，2)确定TRPV6的结构机制抑制，3) 识别门控和调节中潜在相似性和差异的结构元件 TRPV6 和其他 TRP 通道。 TRP 通道是结构功能研究中具有挑战性的目标，因为它们代表大尺寸的多聚体整合膜蛋白，但表达水平通常较低。到为了实现我们的目标，我们将结合使用结构和功能方法，包括现代冷冻技术电子显微镜（冷冻电镜）、X 射线晶体学、蛋白质工程、基于荧光的尺寸排除色谱 (FSEC)、钙成像、荧光光谱和电生理学。我们将表达 TRP通道，它们在真核细胞系中的突变体和遗传变异体，使用不同的膜纯化它们模拟系统，并在存在不同刺激的情况下确定冷冻电镜和晶体结构。我们随后将将新生结构信息与功能数据相结合，以辨别 TRP 通道的分子机制 Ca2+、温度和脂质的门控、抑制和调节。实现我们的目标将显着改善了解 TRP 通道结构和功能，从而为理论提供新的动态模板预测、计算机模拟和新药的化学合成。