Figure 3. Optical studies of ion channel gating.

(a) Stationary noise of single spots, with each curve corresponding to the averaged fitted curves for the indicated conditions (n = 7, paired). The autocorrelation function was obtained for background (gray), capsaicin free (blue), and 3 μM capsaicin (orange). (b) Analysis of fluorescence fluctuations. Superimposed traces of capsaicin (blue) and background (red). The threshold for level one is highlighted (2.5 standard deviations [SD], dotted line). The gray box indicates the zoomed-in portion of the recording presented in the inset below, with the dotted lines indicating background average.. An idealization is shown in black. (c) Semi-log plot of the amplitude versus dwell-time shows a shortening of mean time L₀. (d) Plot summarizing datasets of amplitude versus dwell time at 0 and 3 μM capsaicin (n = 5, paired). Error bars correspond to SEM. (e) Dose–response curve showing changes in the probability of level 1 (P_L1), calculated from non-corrected fluorescence fluctuations, for the two positions tested for the indicated channel types expressing coumarin. (f) Normalized dose–response curves for background subtracted fluorescence fluctuations (orange) and whole-cell electrophysiological response (purple). The electrophysiological response was tested at −70 mV in the presence of the indicated concentrations of capsaicin. Error bars correspond to SEM.

Figure 3—figure supplement 1.

(a) Two lateral views showing the relative position of W426, located on the pre-TM1 region, outside the membrane. The different regions of the monomer are color coded as indicated. The image on the right depicts two subunits and the relative position of the channel with respect to the plasma membrane (diffuse gray). (b) Representative traces obtained from transiently transfected HEK-293T cells subjected to voltage steps from −180 mV to +180 mV in 20 mV increments. Traces correspond to non-stimulated and capsaicin-incubated conditions. (c) Current–voltage relationship for the conditions in b. (d) Raw traces of photon-count recordings in the absence and presence of capsaicin. (e) Capsaicin incubation does not affect photon-count distribution on 426^coummutants. (f) Fluorescence fluctuations at 426^coum. A signal trace is depicted and the background average indicated (red dashed line). The threshold for a single transition is highlighted (2.5 SD, green dashed line). (g) The purple-shaded box on panel f marks the zoomed-in portion of the recording shown here. Lines indicate background average and threshold. Time corresponds to the full zoomed signal inside the box.

Figure 3—figure supplement 2. Distribution of the whole data set obtained from the fluctuation analysis of the fluorescence signal.