Holding potential affects the directionality of Kv1.2 fluorescence deflections but does not affect the overall F-V relationship. (A and B) Representative deflections of Kv1.2 C181 A291C held at −120 (A) and −50 mV (B) in response to 100-ms changes in voltage between −150 and 60 mV. (C) Normalized G-V (closed symbols) and total F-V (open symbols) relationships for Kv1.2 C181 A291C from three different holding potentials (mean ± SEM; n = 3–5), with F-V relationships adjusted to account for upward deflections. From −120 mV (triangles), the G-V had a V_1/2 of −10.7 ± 2.5 mV and a slope factor of 24.8 ± 0.9 mV and corresponding values of −69.5 ± 2.0 mV and 22.7 ± 1.1 mV, respectively, for the F-V. V_1/2 and slope factor data from −80 mV (circles) were −2.2 ± 2.3 mV and 24.6 ± 0.7 mV for the G-V and −69.1 ± 4.5 mV and 20.8 ± 0.8 mV for the F-V. At −50 mV (squares), the V_1/2 and slope factor for the G-V and F-V were 2.6 ± 0.8 mV and 24.2 ± 1.1 mV and −69.9 ± 5.8 mV and 22.6 ± 1.5 mV, respectively. (D) Shaker A359C G-V and F-V relationships from the same three holding potentials as in C (mean ± SEM; n = 7–8). From −120 mV (triangles), the G-V had a V_1/2 of −22.2 ± 2.1 mV and a slope factor of 15.8 ± 1.6 mV and corresponding values of −35.0 ± 4.5 mV and 27.5 ± 1.6 mV, respectively, for the F-V. V_1/2 and slope factor data from −80 mV (circles) were −22.1 ± 1.9 mV and 15.9 ± 1.7 mV for the G-V and −39.8 ± 4.0 mV and 29.1 ± 2.1 mV for the F-V. At −50 mV (squares), the V_1/2 and slope factor for the G-V and F-V were −12.7 ± 1.9 mV and 15.6 ± 1.7 mV and −79.6 ± 4.8 mV and 32.4 ± 1.5 mV, respectively.