Figure 4. Inhibition of TRPM8 activity by PHB-depolymerase PhaZ7 in DRG neurons.

Fluorescence measurements of intracellular Ca²⁺ concentration were performed on DRG neurons with transiently transfected TRPM8 (3 μg) and GFP (0.5 μg) (panel A) (n = 4, _cells= 7), TRPM8 together with the PhaZ7 clone (3 μg) (panel B) (n = 5, n_cells= 10), and mutant S827P (3 μg) (panel C) (n = 3, n_cells= 6). Panels A–C are representatives of single cover slips; total number of experiments and number of neurons are indicated in parentheses. The summaries of averaged cold and menthol responses are represented in panel D (error bars stand for ±s.e.m.). Details of the neuronal transfection are given in supplementary information.

Panel E: PHB signals enhanced in DRG neurons expressing TRPM8. DRG neurons expressing TRPM8/GFP or TRPM8/PhaZ7/GFP were immuno-probed with anti-PHB-IgG and Alexa-546 (red) as secondary antibodies. The transfection conditions are the same as described for the panels A and B. PHB-depolymerase hydrolyzes PHB, which results in reduced signals from plasma membrane and neurites. The upper panel shows PHB staining in DRG neurons transiently expressing TRPM8 and GFP; the two middle panels demonstrate PHB in neurons expressing TRPM8, PhaZ7, and GFP; and the lower panel shows PHB in un-transfected neurons. For immunocytochemical analysis cells were observed with a Zeiss (Oberkochen, Germany) LSM-510 confocal microscope (60X objective), equipped with an Argon laser (488 nm), a Red Diode laser (637 nm), and a Green HeNe laser (543 nm), in the Confocal Imaging Facility of the New Jersey Medical School.