Fluorescence labeling strategies for cell surface expression of TRPV1.

IF 3.3 2区 医学 Q1 PHYSIOLOGY
Journal of General Physiology Pub Date : 2024-10-07 Epub Date: 2024-08-20 DOI:10.1085/jgp.202313523
Taylor M Mott, Grace C Wulffraat, Alex J Eddins, Ryan A Mehl, Eric N Senning
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引用次数: 0

Abstract

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. Here, we present two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb its function. We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry, compatible with a trans-cyclooctene coupled fluorescent dye. Additionally, by inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we can incorporate a fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population. We identified T468 as a rapid labeling site (∼5 min) based on functional and biochemical assays in HEK293T/17 cells. Through adapting linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, we generated a fully functional channel construct, TRPV1exCellHalo, with intact wild-type gating properties. We used TRPV1exCellHalo in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The application of these extracellular label TRPV1 (exCellTRPV1) constructs to track surface localization of the channel will shed significant light on the mechanisms regulating its expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

细胞表面表达 TRPV1 的荧光标记策略。
质膜上离子通道表达的调控是神经元兴奋性的主要决定因素,而确定这种表达的内在机制对于我们了解神经元至关重要。在这里,我们提出了两种正交策略来标记离子通道 TRPV1 的胞外位点,以最小程度地干扰其功能。我们使用琥珀色密码子抑制技术,通过四嗪点击化学方法引入非典型氨基酸(ncAA),并与反式环辛烯偶联荧光染料兼容。此外,通过在 TRPV1 的细胞外环插入环状包覆的 HaloTag(cpHaloTag),我们还可以加入我们选择的荧光染料。ncAA 插入位点的优化是通过筛选 S1 和 S2 跨膜结构域之间的残基位置来实现的。根据在 HEK293T/17 细胞中进行的功能和生化试验,我们确定 T468 为快速标记位点(∼5 分钟)。通过调整链接长度和 cpHaloTag 在 TRPV1 细胞外侧的骨架位置,我们生成了一个具有完整野生型门控特性的全功能通道构建体 TRPV1exCellHalo。我们在单分子实验中使用了 TRPV1exCellHalo 来追踪细胞表面的 TRPV1,并验证了激活后通道流动性降低的研究结果。应用这些细胞外标记 TRPV1(exCellTRPV1)构建体来追踪通道的表面定位,将对其表达的调控机制产生重要启示,并为其他细胞表面受体引入类似修饰提供了通用方案。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
6.00
自引率
10.50%
发文量
88
审稿时长
6-12 weeks
期刊介绍: General physiology is the study of biological mechanisms through analytical investigations, which decipher the molecular and cellular mechanisms underlying biological function at all levels of organization. The mission of Journal of General Physiology (JGP) is to publish mechanistic and quantitative molecular and cellular physiology of the highest quality, to provide a best-in-class author experience, and to nurture future generations of independent researchers. The major emphasis is on physiological problems at the cellular and molecular level.
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