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

瞬时受体电位通道 TRPV3 激活和调节的结构和功能原理。

• 批准号: 10559520
• 负责人: Alexander Sobolevsky
• 金额: $ 58.57万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10559520
• 关键词: Acne Vulgaris; Affinity; Agonist; Alkaloids; Anhydrides; Atopic Dermatitis; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Borates; Calcium; Calmodulin; Camphor; Cells; Chemicals; Collaborations; Combined Modality Therapy; Complex; Coumarins; Cryoelectron Microscopy; Cutaneous; DNA Sequence Alteration; Data; Dermatitis; Detection; Development; Disease; Drug Design; Electrophysiology (science); Endogenous Factors; Esthesia; Eugenol; Fluorescence; Fluorescent Dyes; Functional disorder; Fura-2; Goals; Growth; Guanine; Hair; Heating; High temperature of physical object; Human; Individual; Ion Channel; Ion Channel Gating; Kinetics; Knowledge; Ligands; Lipid Bilayers; Lipids; Maintenance; Malignant neoplasm of lung; Measurement; Mediating; Membrane Lipids; Methodology; Methods; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Mutagenesis; Mutation; Nociception; Olmsted syndrome; Pain; Periodicity; Pharmaceutical Preparations; Phase Transition; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Physiological; Physiological Processes; Plant Extracts; Play; Positioning Attribute; Process; Proteins; Pruritus; Psoriasis; Regulation; Reporting; Research; Role; Rosacea; Signal Transduction; Site-Directed Mutagenesis; Skin; Skin Neoplasms; Skin Physiology; Stimulus; Structural Models; Structure; TRP channel; Techniques; Temperature; Testing; Thermodynamics; Transmembrane Domain; Vanilloid; analog; antagonist; base; citral; colorectal cancer progression; design; disease-causing mutation; farnesyl pyrophosphate; gain of function; inhibitor; inorganic phosphate; isopentenyl pyrophosphate; keratinocyte; mutant; natural product inspired; novel therapeutic intervention; novel therapeutics; overexpression; patch clamp; ratiometric; sensor; skin barrier; skin disorder; small molecule; thermostability; wound healing

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.

项目总结 瞬时受体电位(Trp)通道在许多生理过程中发挥着重要作用，并且 被称为多模式传感器，它在细胞信号中集成了广泛的物理和化学刺激。 TrPV3是Trp通道香草型亚家族的代表，主要在皮肤中表达 角质形成细胞，并与皮肤感觉有关，包括温觉、痛觉和瘙痒 除了维护皮肤屏障、伤口愈合和毛发生长。TRPV3通道功能障碍， 通常是基因突变的结果，与许多人类皮肤病有关，包括 遗传性皮肤病称为Olmsted综合征、牛皮癣、皮肤肿瘤、脱发、皮肤疼痛、瘙痒、瘙痒和 特应性皮炎、酒渣鼻和寻常痤疮。此外，TRPV3的过度表达与 结直肠癌和肺癌的发生和发展。靶向TRPV3用于疾病治疗需要 有关此渠道的结构和功能的详细信息。我们计划研究TRPV3通道 利用不同的生物物理和生化方法相结合的结构和功能。我们的具体目标 主要包括：(1)确定小分子激活TRPV3和致病的分子机制 突变，(2)建立热激活TRPV3的结构基础，以及(3)阐明结构机制 对TRPV3的抑制作用。为了实现我们的目标，我们将使用荧光检测大小排除 层析(FSEC)和热稳定性分析以评估表达、组装、均质性和稳定性 对于TRPV3蛋白，冷冻电子显微镜(Cryo-EM)获得有或没有疾病的TRPV3的结构- 相关突变，在不同温度下以及与不同激活剂、调节剂和 抑制剂，以及与电生理相结合的定点突变，包括单通道 来自平面脂质双层的记录和来自HEK 293细胞的全细胞膜片钳记录，以及比率计量学 钙敏感荧光染料Fura-2 AM测定细胞内钙离子浓度 TRPV3功能，并对我们的结构模型进行关键测试。结合我们的结构和功能结果，我们 将破译TRPV3调节和门控的机制。实现我们的目标将产生重大影响 皮肤生理学和离子通道生物物理学，并将产生新的知识，将有助于结构- 以药物设计为基础，帮助开发新的治疗策略。