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.

项目摘要瞬态受体电势（TRP）通道代表多峰细胞传感器，该传感器整合化学物质，温度，机械应力和膜电压刺激，并将其转换为离子电流以调节我们的视觉，听力，品味，气味和触摸感，并有助于温度和疼痛感知。 TRP通道与包括癌症在内的许多人类疾病的发病机理有关，代表了最热心追求的药物靶标之一。尽管最近在TRP通道结构上取得了成功确定，对它们的遗传多样性，功能和调节的理解仍然远非完整。这种有限的知识代表了基于TRP渠道制定治疗策略的关键障碍调节和对理性药物设计的进展。我们计划研究TRP通道结构和功能结合不同的生物物理和生化方法。我们的具体目的是：1）建立 TRPV6多态性和疾病变异的分子碱基，2）确定TRPV6的结构机制抑制作用和3）确定相似性的基础结构元素以及门控和调节的差异 TRPV6和其他TRP通道。 TRP通道是结构功能研究的挑战，因为它们代表具有通常低表达水平的大尺寸的多聚体积分膜蛋白。到实现我们的目标，我们将结合结构和功能的方法，包括现代的冷冻电子显微镜（冷冻EM），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，其突变体和真核细胞系中的遗传变异，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EM和晶体结构。然后我们会将新生的结构信息与功能数据结合在一起，以识别TRP通道的分子机制 Ca2+，温度和脂质的门控，抑制和调节。实现我们的目标将大大改善了解TRP通道结构和功能，从而为理论带来了新的动态模板预测新药的硅酸拟合和化学合成。