Introduction

Temperature detection and regulation are critical components of various homeostatic and disease-related processes (e.g., thermal adaptation, fever response, inflammation, etc.). Temperature-induced changes in ion channel conductance are one method by which biological systems receive and subsequently quantify thermal information. The quantitative measure of temperature's effect on a given channel is described by the notation Q₁₀, which denotes the fold change in conductance over a span of ten degrees Celsius. Specifically, large temperature effects (Q₁₀ > 4) have been documented for a number of ion channels such as the Shaker (K +), ClC-0 (Cl-), L-type (Ca^{2 +}), and transient receptor potential (TRP) (Ca^{2 +}) channels (1,2,3,4,5). Perhaps most notable among these is a set of TRP family members shown to be involved directly in thermal sensation with cold- (TRPM8, TRPA1), warmth- (TRPV3, TRPV4), and heat-sensing (TRPV1, TRPV2) members (6,7,8,9,10,11,12). Additional members of this family (TRPM2, TRPM4, and TRPM5) were also recently shown to be temperature sensitive (13).

TRPV1 is a well-described cation channel found in nociceptive neurons and present in both C and Aδ fibers, as well as in other neural and non-neural tissues (14). Transgenic mice lacking the TRPV1 gene do not develop inflammatory-mediated hyper-algesia, and profiling of TRPV1 antagonists in multiple preclinical animal models of pain strongly suggests this ion channel plays an important role in pain transmission. At the molecular level, TRPV1 responds to a variety of stimuli including low pH, heat (>42°C), capsaicin, and endogenous ligands such as anandamide (14). This multiplicity of agonists coupled with TRPV1's importance to pain states suggests that TRPV1 may integrate several extracellular signals of inflammatory and neuropathic pain, thus making TRPV1 an attractive target for the development of novel analgesics. As low pH and the presence of inflammatory mediators results in a decrease in TRPV1 gating temperature (15), temperature is a physiologically relevant stimulus for the discovery and development of TRPV1 antagonists as potential drugs (14).

TRPM8 is a member of the long or melastatin family of TRP channels and was originally identified as an up-regulated transcript in a prostate cancer cell line (16). It is expressed primarily in trigeminal and dorsal root ganglia in both A and C fibers, almost exclusively from TRPV1- and TRPA1-expressing neurons (17). TRPM8 is gated by menthol, icilin, and innocuous cold (18–25°C; Q₁₀ = 24). Recent results from studies of TRPM8-deficient mice indicate that TRPM8 is necessary for proper cold detection and cold-induced analgesia, and is at least partly responsible for mediating cold allodynia in neuropathic pain states (18). Thus, it appears that TRPM8 could be a pharmacological target for indications involving cold hyperalgesia or allodynia.

Current TRP channel high-throughput screening (HTS) assays are constructed around the use of either small-molecule agonists or other easily applicable stimuli [e.g., capsaicin or protons (low pH) for TRPV1] to gate the channel and induce Ca^{2 +} influx into cells. In these assays, cells were first loaded with fluorescent Ca^{2 +}-sensitive dyes followed by treatment with test compounds (antagonists). Cells were then exposed to an agonist and Ca^{2 +} influx was assessed by measuring changes in intracellular fluorescence with platforms such as Flexstation or FLIPR (Molecular Devices, Sunnyvale, CA, USA). Interestingly, to the best of our knowledge, there have been no published reports of microplate-based assays using continuous temperature changes to trigger TRP channel opening. However, the induction of stepwise, temperature-mediated channel activation using buffers of differing temperature has been reported (19,20). While electrophysiology is another technology that can be used to screen compounds using capsaicin and low pH, it has a much lower throughput when temperature is used as the channel-opening modality (21). In the current study, we describe the use of a standard real-time PCR machine to develop a cell-based, functional assay employing temperature as an agonist for ion channel gating. We demonstrate this assay's potential for compound screening and its use in the analysis of small molecules targeting TRPV1. Furthermore, we subsequently describe the application of the assay to another temperature-sensitive TRP channel, the cold sensor TRPM8.

Materials and methods

Reagents

Tissue culture reagents, 5′-iodo resiniferatoxin (I-RTX), Fluo-4 AM, and capsazepine were purchased from Mediatech (Manassas, VA, USA), Tocris-Cookson (Ellisville, MO, USA), Invitrogen (Carlsbad, CA, USA), and Sigma-Aldrich (St. Louis, MO, USA), respectively. Other chemical compounds were synthesized in-house.