Figure 1. Representative fluorescence intensity traces from Fluo-3-loaded cells measuring real-time intracellular Ca²⁺ responses to TRPM8 agonist.

Relative fluorescence intensity units (RFU) are plotted versus time (s). (a) HEK-293 cells expressing human TRPM8 prepared in the presence of increasing concentrations of probenecid (A: 0.13 mM, B: 1.3 mM, C: 2.5 mM) treated with 40 nM WS 12 (injected at first arrow), then 2 μM A23187 (arrow with asterisk, indicated for first trace). Exposure to probenecid suppressed the response to WS 12. (b) Example Ca²⁺ transient traces showing dose-dependent WS 12 activation of TRPM8 SV 762,763 EL double residue mutant expressed in HEK-293 cells (A: 1 μM, B: 100 nM, C: 30 nM WS 12). (c) The Ca²⁺ transient response to 125 nM WS 12 in SH-SY5Y cells transfected with TRPM8 was similar in magnitude to the response to 2 nM bradykinin-mediated activation of endogenous bradykinin receptors, (d) (e) Dose–response curves derived from measurements of peak intracellular Ca²⁺ fluorescence in HEK-293 and SH-SY5Y cell clones expressing TRPM8 constructs. Estimation of EC₅₀ for WS 12 (mean±S.E.M., n=3 independent experiments each performed in triplicate). Mean EC₅₀ values for reference sequence and mutant TRPM8 were not statistically significant in either cell line. (f) Estimated IC₅₀ values for TRPM8 antagonist AMTB determined using a constant dose of 150 nM WS 12, (mean±S.E.M., n=3). Mean IC₅₀ values for wild-type and mutant TRPM8 were not statistically significant in either cell type.