瞬时受体电位通道TRPV6的表达、纯化及结晶

Expression, Purification, and Crystallization of the Transient Receptor Potential Channel TRPV6.

作者信息

Singh Appu K, McGoldrick Luke L, Sobolevsky Alexander I

机构信息

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

Integrated Program in Cellular, Molecular and Biomedical Studies, Columbia University, New York, NY, USA.

出版信息

Methods Mol Biol. 2019;1987:23-37. doi: 10.1007/978-1-4939-9446-5_2.

DOI:10.1007/978-1-4939-9446-5_2

PMID:31028671

Abstract

Transient receptor potential (TRP) channels are polymodal sensory transducers that respond to chemicals, temperature, mechanical stress, and membrane voltage and are involved in vision, taste, olfaction, hearing, touch, thermal perception, and nociception. TRP channels are implicated in numerous devastating diseases, including various forms of cancer, and represent important drug targets. The large sizes, low expression levels, and conformational dynamics of TRP channels make them challenging targets for structural biology. Here, we present the methodology used in structural studies of TRPV6, a TRP channel that is highly selective for calcium and mediates Ca uptake in epithelial tissues. We provide a protocol for the expression, purification, and crystallization of TRPV6. Similar approaches can be used to determine crystal structures of other membrane proteins, including different members of the TRP channel family.

摘要

瞬时受体电位（TRP）通道是多模态感觉转导器，可对化学物质、温度、机械应力和膜电压作出反应，并参与视觉、味觉、嗅觉、听觉、触觉、热感知和痛觉感受。TRP通道与多种严重疾病有关，包括各种形式的癌症，是重要的药物靶点。TRP通道的大尺寸、低表达水平和构象动力学使其成为结构生物学具有挑战性的靶点。在这里，我们介绍了TRPV6结构研究中使用的方法，TRPV6是一种对钙具有高度选择性并介导上皮组织中钙摄取的TRP通道。我们提供了TRPV6表达、纯化和结晶的方案。类似的方法可用于确定其他膜蛋白的晶体结构，包括TRP通道家族的不同成员。