Figure 1.
Inhibition of TRPM8 currents by scPPX1 in whole-cell patch clamp.
Upper panels: Whole-cell patch clamp measurements of menthol-induced currents were performed at −60 mV in the whole-cell configuration on HEK cells expressing TRPM8, in nominally Ca2+-free solution (NCF), to avoid desensitization. Menthol pulses (500 µM) were applied in the first 3–5 min after establishment of whole-cell configuration: HEK-293 cells were transiently transfected with TRPM8 (0.4 µg) and co-transfected with GFP clone (0.2 µg) to allow detection of transfected cells. Panel A: the control. Panel B: the pipette solution was supplemented with 2.3 µg/ml scPPX1. Midle panels: Whole-cell patch clamp was performed on HEK-293 TRPM8 stable cell line, which was transiently transfected with GFP (0.2 µg) alone (panel D) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg) (panel E). The summaries are shown in panel F. The protocol of experiment is the same as for the measurements in the upper panel. Lower panels: Current/Voltage relationships of TRPM8 channels obtained in whole-cell patch clamp performed at −100 +100 mV voltage ramps for HEK-293 TRPM8 stable cell line, which was transiently transfected with GFP (0.2 µg) alone (panel G) or with scPPX1 clone (0.4 µg) and GFP (0.2 µg) (panel H). The summaries are shown in panel I at −100 and +100 mV.
Figure 2.
Inhibition of TRPM8 activity by scPPX1 in intracellular Ca2+ measurements.
Upper panels: Fluorescence measurements of intracellular Ca2+ concentration were performed on HEK-293 TRPM8 stable cell lines with transiently transfected GFP (0.2 µg) alone (panel A) or together with the scPPX1 clone (0.4 µg) (panel B). The summaries of averaged menthol responses are represented in panel C. Lower panels: Fluorescence measurements of intracellular Ca2+ signals were performed on F-11 neuronal cells with transiently transfected TRPM8 (0.4 µg) and GFP (0.2 µg) (panel D) or together with the scPPX1 clone (0.4 µg) (panel E). The summaries of averaged menthol responses are represented in panel F.
Figure 3.
Western blots of TRPM8 protein derived from expression in HEK-293 cell lines.
TRPM8 protein samples were separated on a 10% SDS-PAGE and blotted on nitrocellulose membranes overnight in the presence of CAPS buffer (pH 11.1). Immunodetection was revealed by chemiluminescence. Lanes 1–3 probed with anti-Myc-IgG: Lane 1 – plasma membrane fractions of HEK-293 cells not expressing TRPM8; Lane 2 – plasma membrane extracts of cells stably expressing TRPM8; Lane 3 – TRPM8 protein purified on Sephacryl-300 gel-filtration chromatography. Lane 4 – Coomassie blue staining of purified TRPM8. Samples were heated for 5 min. at 70°C before loading.
Figure 4.
A. Detection of polyP associated with the TRPM8 protein. TRPM8 was separated on native PAGE to preserve its migration in the tetrameric form. Lane 1 – standards ladder (The High-Mark Pre-stained High Molecular Weight Protein Standards, Invitrogen); Lane 2 – purified TRPM8 sample with o-toluidine blue stain of native PAGE gel; Lane 3 – o-toluidine blue stain of native PAGE gel of the same TRPM8 sample treated with 1 µl scPPX1 (2 µg/ml) for 3 h. before loading: Lane 4 and 5 are lanes 2 and 3 re-stained with Coomassie blue. B. Detection of PHB in TRPM8 in Western blot. Lane 1 – purified TRPM8 protein detected with antiMyc_IgG; Lane 2 – Western blot of purified TRPM8 probed with anti-PHB-IgG. Samples were heated for 5 min. at 70°C before loading.
Figure 5.
Activation of TRPM8 channels in Planar Lipid Bilayers by menthol and PtdIns(4,5)P2.
Representative single-channel current recordings of TRPM8 channels incorporated in planar lipid bilayers formed from POPC/POPE (3∶1) in n-decane, between symmetric bathing solutions of 150 mM KCl, 0.2 mM MgCl2 in 20 mM Hepes buffer, pH 7.4 at 22°C. 0.2–0.5 µl of 0.2 µg/ml TRPM8 protein (isolated from the plasma membrane of HEK-293 cells stably expressing TRPM8) was incorporated in POPC/POPE micelles, which were added to the cis compartment (ground). Clamping potential was +60 mV. Data were filtered at 50 Hz. Upper and lower traces consist of three segments with additions of components as indicated in the figure: 2 µM of diC8 PtdIns(4,5)P2 and 500 µM of menthol were added to both compartments. The current recordings are representative of a total of 22 independent experiments for the upper traces and 12 independent experiments for the lower traces.
Figure 6.
A. Representative current/voltage relationship of TRPM8: Channels were incorporated in planar lipid bilayers of synthetic POPC, POPE (3∶1) in the presence of diC16 PtdIns(4,5)P2. Experimental conditions are the same as described in the legend to Fig. 5. TRPM8 channels were stimulated with the application of 500 µM of menthol. The dashed line corresponds to the mean conductance of fully open channels, working in inward direction, this state is rarely observed due to the low open probability of this subconductance level. B: Representative current traces and all points' histograms of outward (upper) and inward (lower) currents of TRPM8 channels with clamping potentials were +60 mV and −60 mV, respectively. Experimental conditions are the same as in the legend to figure 6A. C: Open probability of TRPM8 channels operating in inward and outward directions measured at +100 mV and −100 mV. Data were analyzed from a total of 9 experiments. D: Menthol dose response of the open probability of TRPM8. Demonstrated Po values were obtained at 100 mV. Data were analyzed from a total of 36 experimens.
Figure 7.
Activation of TRPM8 channels in Planar Lipid Bilayers by cold.
Representative current traces of TRPM8 activated by lowering the temperature from 23 to 16°C in planar lipid bilayers: Channels were incorporated in planar lipid bilayers of synthetic POPC, POPE (3∶1) in presence of diC16 PtdIns(4,5)P2. Experimental conditions are the same as described in the legend to Fig. 6. Channels were inserted cis at 23°C and the temperature was then lowered to 16°C at ∼1 degree per min. Upper trace: TRPM8 activity at 23°C; lower trace: TRPM8 channel activity at 16°C (representative of 12 independent experiments). The temperature of the chambers was controlled by pyroelectric controller (see Experimental Procedures). The temperature in the cis bath (ground) was read directly using a thermoelectric junction thermometer, which also served as a point of reference for the pyroelectric controller. Data were filtered at 50 Hz. Clamping potential was −60 mV.
Figure 8.
Voltage-dependence of TRPM8 before and after the treatment with scPPX1.
A: Representative current traces recordings obtained at −150 +150 mV voltage ramps before and after the treatment with polyphosphatase in a time course at the beginning of 3rd, 10th, 18th, 28th and 33rd minutes. B: The changes in open probability obtained at different voltages in gap free recordings for TRPM8 alone (▪) or after the treatment with scPPX1 for the following intervals of time: 5–7 min (♦), 9–11 min (Δ), 14–16 min (▾), 20–23 min (◊), and 28–32 min (•). Data were analyzed from overall of 16 experiments.
Figure 9.
Reduction of TRPM8 channel conductance by exopolyphosphatase scPPX1.
A: Representative single-channel current recordings of TRPM8 channels: upper traces – TRPM8 channels recordings before treatment with scPPX1; middle traces – TRPM8 channel recording 15 minutes later after the addition of scPPX1 (2 µg); lower traces – TRPM8 channel recordings after 30 minutes of addition of scPPX1. Clamping potential was +100 mV. Data were filtered at 50 Hz. B: Symbols (▪) (n = 5) correspond to the mean conductance values of scPPX1 treated TRPM8 channels, where 2 µM of scPPX1 were added to the internal side of the channel; (○) (n = 8) mean conductance of control, untreated channels. Experimental conditions are the same as described in the legend to Fig. 6.