Figure 5. Depletion of PI(4,5)P2 by the rapamycin-induced phosphoinositide phosphatase gives less inhibition in TRPV1-R721A channels than in TRPV1-WT. (A) Representative experiments of simultaneous electrophysiological and confocal imaging recordings on HEK 293T/17 cells expressing TRPV1-WT (left) or TRPV1-R721A (right) with the chemically inducible phosphatase expression system. The PH-YFP fluorescence images are shown, and the numbers illustrate when in the current time course below the images were taken. The graphs show the perforated patch whole-cell currents at −60 mV (left axis, black circles) overlaid with the simultaneous fluorescence measurement in a region of interest of the cytosol in the patched cell (right axis, yellow circles). The bars above the graph represent when additions were made. The red to blue bar below the data are a cartoon to help the reader visualize how PI(4,5)P2 and PI(4)P levels in the plasma membrane are changing during the experiments, based on the observed translocation of the PH-YFP fluorescent PI(4,5)P2 probe. Currents were activated by 1 µM capsaicin (black bars) in nominally Ca2+-free solution. Once current activation reached steady-state, 1 µM rapamycin (green bars) was added to induce phosphatase localization to the plasma membrane and therefore dephosphorylation of plasma membrane PI(4,5)P2, its depletion tracked by the movement of PH-YFP. (B) Data representation in the box plots is as in Fig. 3. (left) Box plot of the capsaicin-activated current left after complete PH-YFP translocation to the cytosol (representing depletion of plasma membrane PI(4,5)P2) for TRPV1-WT and TRPV1-R721A. The asterisk indicates a statistically significant difference (P < 0.001). For both TRPV1-WT and TRPV1-721A, seven cells were included in the analysis. (right) Box plot comparing PH-YFP translocation in TRPV1-WT and TRPV1-R721A cells. Translocation index was calculated as cytosolic fluorescence after PH-YFP translocation has stopped, divided by the initial cytosolic fluorescence. For both TRPV1-WT and TRPV1-721A, seven cells were included in the analysis. The meaning of the boxes and whiskers is the same as in Fig. 4.
Figure 5.

Depletion of PI(4,5)P2 by the rapamycin-induced phosphoinositide phosphatase gives less inhibition in TRPV1-R721A channels than in TRPV1-WT. (A) Representative experiments of simultaneous electrophysiological and confocal imaging recordings on HEK 293T/17 cells expressing TRPV1-WT (left) or TRPV1-R721A (right) with the chemically inducible phosphatase expression system. The PH-YFP fluorescence images are shown, and the numbers illustrate when in the current time course below the images were taken. The graphs show the perforated patch whole-cell currents at −60 mV (left axis, black circles) overlaid with the simultaneous fluorescence measurement in a region of interest of the cytosol in the patched cell (right axis, yellow circles). The bars above the graph represent when additions were made. The red to blue bar below the data are a cartoon to help the reader visualize how PI(4,5)P2 and PI(4)P levels in the plasma membrane are changing during the experiments, based on the observed translocation of the PH-YFP fluorescent PI(4,5)P2 probe. Currents were activated by 1 µM capsaicin (black bars) in nominally Ca2+-free solution. Once current activation reached steady-state, 1 µM rapamycin (green bars) was added to induce phosphatase localization to the plasma membrane and therefore dephosphorylation of plasma membrane PI(4,5)P2, its depletion tracked by the movement of PH-YFP. (B) Data representation in the box plots is as in Fig. 3. (left) Box plot of the capsaicin-activated current left after complete PH-YFP translocation to the cytosol (representing depletion of plasma membrane PI(4,5)P2) for TRPV1-WT and TRPV1-R721A. The asterisk indicates a statistically significant difference (P < 0.001). For both TRPV1-WT and TRPV1-721A, seven cells were included in the analysis. (right) Box plot comparing PH-YFP translocation in TRPV1-WT and TRPV1-R721A cells. Translocation index was calculated as cytosolic fluorescence after PH-YFP translocation has stopped, divided by the initial cytosolic fluorescence. For both TRPV1-WT and TRPV1-721A, seven cells were included in the analysis. The meaning of the boxes and whiskers is the same as in Fig. 4.

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