Structural and functional principles of activation and regulation of the transient receptor potential channel TRPV3.

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PROJECT SUMMARY Transient receptor potential (TRP) channels play important roles in numerous physiological processes and are known as polymodal sensors that integrate a wide range of physical and chemical stimuli in cellular signaling. TRPV3, a representative of the vanilloid subfamily of TRP channels, is predominantly expressed in skin keratinocytes and implicated in cutaneous sensation, including thermo-sensation, nociception, and itch, in addition to maintenance of the skin barrier, wound healing, and hair growth. The dysfunction of TRPV3 channels, often a result of genetic mutations, is associated with numerous human skin diseases, including a genodermatosis known as Olmsted syndrome, psoriasis, skin tumors, hair loss, cutaneous pain, itch, pruritic and atopic dermatitis, rosacea, and acne vulgaris. Furthermore, overexpression of TRPV3 is implicated in the development and progression of colorectal and lung cancer. Targeting TRPV3 for disease treatment requires detailed information about the structure and function of this channel. We plan to study the TRPV3 channel structure and function using a combination of different biophysical and biochemical methods. Our specific aims are: (1) determine the molecular mechanisms of TRPV3 activation by small molecules and disease-causing mutations, (2) establish structural bases of TRPV3 activation by heat, and (3) elucidate structural mechanisms of TRPV3 inhibition. To achieve our goals, we will use the Fluorescence-detection Size Exclusion Chromatography (FSEC) and thermostability assays to assess expression, assembly, homogeneity and stability of the TRPV3 protein, cryo-electron microscopy (cryo-EM) to obtain structures of TRPV3 with or without disease- associated mutations, at different temperatures and in complex with different activators, modulators and inhibitors, as well as site-directed mutagenesis combined with electrophysiology, including single-channel recordings from planar lipid bilayers and whole-cell patch-clamp recordings from HEK 293 cells, and ratiometric measurements of intracellular Ca2+ concentration using calcium-sensitive fluorescent dye Fura-2 AM to assess TRPV3 function and to critically test our structural models. Combining our structural and functional results, we will decipher the mechanisms of TRPV3 regulation and gating. Achieving our aims will have a significant impact on skin physiology and ion channel biophysics and will generate new knowledge that will assist in structure- based drug design and help the development of new therapeutic strategies.