Structure and function of Transient Receptor Potential Channels

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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.