Figure 3.

Inhibition of TRPM8 activity by PHB-depolymerase, PhaZ7, in intracellular Ca²⁺-measurements

Panels A–C: Fluorescence measurements of intracellular Ca²⁺ concentration were performed on HEK-293 TRPM8 stable cell lines with transiently transfected either with GFP (0.2 μg) alone (panel A) (n = 7, n_cells= 209) or together with the PhaZ7 clone (0.7 μg) (n = 15, n_cells= 277) and the S136A-PhaZ7 mutant – inactive enzyme (0.7 μg) (n = 3, n_cells= 61) (panel B). The summaries of averaged cold and menthol responses with GFP-positive cells are represented in panel C (p < 0.0005; error bars stand for ±s.e.m.).

Panels D–K: Fluorescence measurements of intracellular Ca²⁺ concentration were performed on HEK-293 transiently transfected with TRPM8 (0.7 μg) and GFP (0.2 μg) panel D (n = 7, n_cells=71). Further, TRPM8-expressing cells were treated for 1 h with an inactive form of PHB-depolymerase – S136A PhaZ7 (panel E) (n = 3, n_cells= 26) and wild-type PhaZ7 (panel F) (n = 3, n_cells= 40). Ca²⁺ signals obtained from the HEK cells transiently expressing TRPM8 mutants: L825G (panel G) (n = 5, n_cells= 83), V830G (panel H) (n = 6, n_cells= 90), R829E (panel I) (n = 5, n_cells= 39), and Y826G (panel J) (n = 5, n_cells= 37). Panels A–J are representative of single cover slips; the total number of measurements and the total number of cells for each variety are indicated in parentheses.

The summaries of averaged cold, menthol, and icilin responses are represented in panel K (error bars stand for ±s.e.m.).