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. 2017 Dec 5;6:e28626. doi: 10.7554/eLife.28626

Figure 1. Functional expression of coumarin-containing TRPV1 channels at the membrane of HEK-293T cells.

(a) Top-down and lateral view of the pore region of rTRPV1 tetramers. Dashed lines denote the positions of putative gates, a canonical gate at I679 and a secondary constriction within the pore at Y671 (green shade). The different subunits are shown in different colors. (b) Epifluorescence images obtained from transiently transfected HEK-293T cells. Cells were co-transfected with wild-type rTRPV1-YPF and with rTRPV1-Y671coum. The scale bar corresponds to 10 µm. Images correspond to the average of 50 frames. (c) Representative traces obtained from transiently transfected HEK-293T cells subjected to voltage steps from −180 mV to +180 mV, in 20 mV increments, for the three different conditions indicated. No TRPV1 currents were seen from expressed Y671TAG channels co-expressed with the coumarin Rs and tRNA but lacking the supplemented amino acid in the culture media. (d) Conductance-voltage relation for the wild-type rTRPV1 (black) and the two different coumarin-containing channels (TRPV1-coumarin alone in red; 1xTRPV1coumarin +7xTRPV1wt in blue). Curves were fitted to a Boltzmann distribution of the form I = Imax/(1 + exp([zF(V-V0.5)/RT]), where z is the voltage dependency, V0.5 is the half-activation voltage, and Imax is the maximum current. Each curve represents the average of at least six different experiments performed at 20°C. Error bars correspond to standard errors of the mean (SEM). The structure of coumarin and tyrosine are depicted in wireframe. (e) Ten thousand frames were averaged and a threshold was set on the averaged image in order to define regions of interest (arrow indicates ROI). The scale bar corresponds to 5 µm. (f) The photon count was extracted from the original set of frames. Capsaicin incubation promotes a higher photon count distribution.

Figure 1.

Figure 1—figure supplement 1. Two composites of epifluorescence images obtained from transiently transfected HEK-293T cells.

Figure 1—figure supplement 1.

Cells were co-transfected with wild-type rTRPV1-YPF and rTRPV1-Y671coum. Scale bars correspond to 10 µm. Images correspond to the average of 40 frames (upper set) and 50 frames (lower set).

Figure 1—figure supplement 2. Capsaicin induces an increase in the photon count.

Figure 1—figure supplement 2.

(a) 10, individual frames were taken with the 64 × 32 single-photon avalanche diode (SPAD) camera. (b) Images were averaged to identify cell area and regions of interest (ROIs). Scale bar represents 5 μm. (c) Raw traces of photon count recordings in the absence and presence of capsaicin. (d) Zoomed raw traces comparing background and coumarin-stimulated signals.