Fluorescence from spHCN R332C channels labeled with ALEXA-488 and MTS-TAMRA report on mode-shift development. (A) Representative simultaneous current and ΔF recordings for an oocyte expressing spHCNR332C labeled with ALEXA-488 and superfused in 100K, 100K+Cs, and 100Na solutions in response to increasing prepulse steps to −160 mV, incremented by 80 ms from an initial 8-ms wide pulse. The traces are aligned at the prepulse OFF transition to +40 mV. Insets show respective normalized tail currents at +40 mV plotted on an enlarged time scale. To resolve the tail currents, capacitive transients were eliminated by subtracting the response to the 8-ms wide pulse to −160 mV. ΔF shown as percentage of background fluorescence. Dotted lines show that ΔF increases for superfusing with 100K+Cs and 100Na relative to 100K. The traces were signal averaged fourfold to improve the signal-to-noise ratio. ΔF shown as a percentage of background fluorescence. (B) Time for tail current to decay to 50% of its initial amplitude () combining data with 8- and 80-ms pulse increments and shown relative to the tail response 16-ms pulse for three superfusion conditions plotted as a function of the prepulse width. Data shown as mean ± SEM, n = 10 (100K); 6 (100K+Cs); 6 (100Na). Points fitted with a single exponential function with time constants: 112 ± 12 ms (100K); 208 ± 25 ms (100K+Cs) and 490 ± 81 ms (100Na) (mean ± SE). (C) Representative simultaneous current and ΔF recordings for an oocyte expressing spHCNR332C labeled with MTS-TAMRA, same conditions as in A. Insets show normalized tail currents as in A. ΔF shown as percentage of background fluorescence. Dotted lines show that ΔF increases for 100K+Cs and 100Na relative to the 100K condition. (D) Time for tail current to decay to 50% of its initial amplitude () combining data with 8- and 80-ms pulse increments as in B. Data shown as mean ± SEM, n = 9 (100K); 7 (100K+Cs); 6 (100Na). Points fitted with a single exponential function with time constants: 108 ± 7 ms (100K); 173 ± 15 ms (100K+Cs) and 105 ± 7 ms (100Na) (mean ± SE). (E) Correlations at −160 mV between Iact (black trace) and the slow component of ALEXA-488 ΔF from the same cell as in A (blue trace), for superfusion conditions as indicated. Iact is shown inverted, and Iact and ΔF are normalized to corresponding values at 488 ms. Relative tail delay () data from B was overlaid on the same abscissa according to the prepulse width so that the values at 16 and 488 ms match ΔF at these time points. Insets with ΔF trace expanded from 0 to100 ms, show that for 100K+Cs and 100Na at short prepulse periods (up to 88 ms) there was an improved match between the time course of ΔF and . Scale bars for insets: vertical 5 ms, horizontal 50 ms. (F) Correlations at −160 mV between Iact (black trace) for the slow component of MTS-TAMRA ΔF (green trace) from the same cell as in C, for superfusion conditions as indicated, plotted against time. Iact was inverted and ΔF normalized as in E. Inset for 100K superfusion shows an expanded view to 88 ms obtained from the same cell using the 8-ms time increment protocol with Iact and ΔF normalized to values at 88 ms time point. Scale bar = 40 ms. Relative tail delay () data from D overlaid on the same abscissa according to the prepulse width according to the prepulse width so that the values at 16 and 488 ms match ΔF at these time points.