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。

基本信息

批准号：
10365295
负责人：
Alexander Sobolevsky
金额：
$ 59.96万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/10365295
关键词：
Acne Vulgaris Affinity Agonist Alkaloids Anhydrides Atopic Dermatitis Binding Binding Sites Biochemical Biological Assay Biophysics Borates Calcium Calmodulin Camphor Cells Chemicals Colorectal 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 Lung Maintenance Malignant neoplasm of lung Measurement Mediating Membrane Lipids Methodology Methods Molecular Molecular Conformation Molecular Sieve Chromatography Mutagenesis Mutation Natural Products 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 isopentenyl pyrophosphate keratinocyte mutant novel therapeutic intervention novel therapeutics overexpression patch clamp ratiometric receptor sensor skin barrier skin disorder small molecule thermostability wound healing

项目摘要

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.

项目概要瞬时受体电位 (TRP) 通道在许多生理过程中发挥着重要作用，被称为多模态传感器，在细胞信号传导中集成了广泛的物理和化学刺激。 TRPV3 是 TRP 通道香草酸亚家族的代表，主要在皮肤中表达角质形成细胞并与皮肤感觉有关，包括热感觉、伤害感受和瘙痒，除了维护皮肤屏障、伤口愈合和头发生长之外。 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（称为奥姆斯特德综合征）、牛皮癣、皮肤肿瘤、脱发、皮肤疼痛、瘙痒、瘙痒和特应性皮炎、红斑痤疮和寻常痤疮。此外，TRPV3 的过度表达与结直肠癌和肺癌的发生和进展。针对 TRPV3 进行疾病治疗需要有关该通道的结构和功能的详细信息。我们计划研究TRPV3通道结合不同的生物物理和生化方法来实现结构和功能。我们的具体目标是：（1）确定小分子激活TRPV3及致病的分子机制突变，(2) 建立 TRPV3 热激活的结构基础，(3) 阐明结构机制 TRPV3 抑制。为了实现我们的目标，我们将使用荧光检测尺寸排除通过色谱 (FSEC) 和热稳定性测定来评估表达、组装、均质性和稳定性 TRPV3 蛋白，冷冻电子显微镜 (cryo-EM) 以获得有或无疾病的 TRPV3 结构- 相关的突变，在不同的温度下并与不同的激活剂、调节剂和复合物抑制剂，以及与电生理学相结合的定点诱变，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，以及比率测量使用钙敏感荧光染料 Fura-2 AM 测量细胞内 Ca2+ 浓度以评估 TRPV3 功能并严格测试我们的结构模型。结合我们的结构和功能结果，我们将破译 TRPV3 调控和门控机制。实现我们的目标将产生重大影响关于皮肤生理学和离子通道生物物理学，并将产生有助于结构的新知识基于药物设计并帮助开发新的治疗策略。