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. 2022 Apr 22;119(18):e2118152119. doi: 10.1073/pnas.2118152119

Fig. 3.

Fig. 3.

Biophysical properties of sTRP1 channel. (A) Representative whole-cell currents of sTRP1-expressing HEK 293T cells elicited by a family of voltage pulses ranging from −100 mV to 200 mV with a 20-mV increment as indicated at upper left, in the presence of normal bath solution (control), 0.2 mM citronellal, or 0.5 mM citronellal. Note, the channels were presensitized by repeated applications of 2 mM citronellal. Vh = −60 mV. (B) G-V relationships derived from the recordings shown in A. Solid lines correspond to fits with Boltzmann function, yielding V1/2 = 134.0 ± 3.4 mV and κ = 36.1 ± 3.2, gating charge = 0.71 ± 0.06 for control (n = 8); V1/2 = 94.6 ± 1.8 mV and κ = 38.6 ± 1.5, gating charge = 0.66 ± 0.02 for 0.2 mM citronellal (n = 8), and V1/2 = 62.8 ± 2.9 mV and κ = 30.5 ± 2.7, gating charge = 0.84 ± 0.07 for 0.5 mM citronellal (n = 5). (C) Single-channel currents of sTRP1 recorded from outside-out membrane patches of HEK 293T cells evoked by 0.2 mM citronellal at the indicated holding potentials after sensitization. Sensitization was induced with 2 mM citronellal. Dotted lines indicate the closed channel state. (D) Plot of unitary current amplitudes versus voltages. Note that the unitary currents were determined by fitting all-point histograms with Gaussians. Unitary conductance measured by fitting a linear function was 152.9 ± 1.5 pS (n = 10). (E) Current-voltage relations. Currents were elicited with 100-ms test pulses ranging from −100 mV to +100 mV at an increment of 20 mV for the same cell exposed to different extracellular solutions containing 0.5 mM citronellal and varying cations as indicated (n = 5). Pipette solutions contained 140 mM NaCl. (F) [Ca2+]i increases elicited by different agonists. Responses of sTRP1-expressing HEK 293T cells to 2 mM citronellal or 2 mM citronellol measured by GCaMP6m fluorescence with 1.8 mM extracellular Ca2+. Color bar indicates the calibration of intracellular calcium concentration, [Ca2+]i. Activation of sTRP1 resulted in the rise of [Ca2+]i. Images showing the levels of [Ca2+]i in HEK 293T cells expressing sTRP1 and GCaMP6m both at rest and in response to citronellal and citronellol, respectively. (Scale bar, 100 μm.) (G) Time courses of the relative change of fluorescence were plotted from the images shown in F. Error bars indicate SEM.