Institute of Biomolecular Chemistry (ICB)-National Research Council (CNR), Pozzuoli, NA, Italy.
Epitech Group SpA, Padova, Italy.
Methods Mol Biol. 2023;2576:119-131. doi: 10.1007/978-1-0716-2728-0_9.
The transient receptor potential vanilloid 1 ion channel (TRPV1) is a ligand-gated nonselective calcium-permeant cation channel involved in the detection of a wide variety of chemical and physical noxious stimuli, ranging from exogenous and endogenous ligands to noxious heat (>42 °C) and low pH (pH < 5.2). Due to its central role in pain and hyperalgesia, TRPV1 is considered a relevant therapeutic target for the development of analgesic and anti-inflammatory drugs potentially useful to relieve chronic, neuropathic, and inflammatory pain and to treat disorders such as inflammatory bowel disease. In this view, the availability of in vitro assays for the screening of novel TRPV1 modulators is highly desirable. Since TRPV1 activation leads to an increase in the intracellular calcium (Ca) levels, the use of Ca fluorescent indicators represent a valuable and sensitive tool for monitoring such intracellular changes. In this chapter, we describe methods for recording and monitoring Ca signals through the fluorescent indicators Fluo-4 acetoxymethyl (AM) and Fura-2 AM in HEK-293 cells transfected with TRPV1 or other thermoTRP channels.
瞬时受体电位香草酸 1 型离子通道（TRPV1）是一种配体门控非选择性钙通透阳离子通道，参与检测多种化学和物理有害刺激，包括外源性和内源性配体、有害热（>42°C）和低 pH 值（pH<5.2）。由于 TRPV1 在疼痛和痛觉过敏中起核心作用，因此被认为是开发潜在用于缓解慢性、神经性和炎症性疼痛以及治疗炎症性肠病等疾病的镇痛和抗炎药物的相关治疗靶点。鉴于此，筛选新型 TRPV1 调节剂的体外检测方法非常重要。由于 TRPV1 的激活会导致细胞内钙（Ca）水平升高，因此使用 Ca 荧光指示剂是监测这种细胞内变化的一种有价值且敏感的工具。在本章中，我们描述了在转染 TRPV1 或其他热 TRP 通道的 HEK-293 细胞中，通过荧光指示剂 Fluo-4 乙酰甲酯（AM）和 Fura-2 AM 记录和监测 Ca 信号的方法。
