Kv1.2 A291C voltage-dependent fluorescence is well characterized by a double exponential function. (A) Fit of the Kv1.2 A291C fluorescence signal at 60 mV to a double exponential shows that ∼40% of the signal amplitude results from a fast movement, with a time constant of 1.3 ms. The slow phase, comprising 60% of the total signal, is slower by an order of magnitude, 23.9 ms in this example. (B) Mean ± SEM time constants of the fast and slow fluorescence signal components (n = 14–20) compared with time constants of ionic current activation fit from ∼50% of maximal activation. (C) Normalized F-V relationships of the fast and slow components of Kv1.2 A291C fluorescence normalized and plotted alongside the G-V relationship (n = 11). The normalized fast phase, fit to a Boltzmann distribution, had a V_1/2 and slope factor of −39.5 ± 2.0 mV and 15.6 ± 1.0 mV. The voltage dependence of the slow phase was best fit with a double Boltzmann function, with the first component having a V_1/2 and k of −73.9 ± 1.4 mV and 12.0 ± 0.5 mV (amplitude = 88.7 ± 2.3%) followed by a second component (11.2 ± 2.5%) with respective V_1/2 and slope factors of 44.3 ± 4.2 mV and 11.6 ± 3.0 mV. (D) Holding potential–dependent separation of the fast and slow fluorescence components. Representative fluorescence traces are shown for Kv1.2 C181 A291C channels depolarized to −30 mV (gray traces) or 60 mV (black traces) from holding potentials (HP) of either −80 or −50 mV as labeled. The two vertical lines to the left of the fluorescence records show the contributions of fast and slow quenching components for depolarizations to 60 mV.