Fluorescence-Based Assay for TRPV1 Channels.

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.