(A) Quenching of Anap fluorescence measured using PCF by different concentrations of Co2+ with or without dihistidines and in the absence and presence of 2 mM ATP/Mg2+ at the resting holding voltage of −100 mV. (B) tmFRET efficiency as a function of Co2+ concentration in the absence and presence of ATP/Mg2+ as described. The smooth curves are fits of the Langmuir isotherm, Apparent FRETeff. = FRETeff [Co2+] / (K1/2 + [Co2+]), with the following parameters: FRETeff = 0.46, K1/2 = 48.0 µM with ATP (red) and FRETeff = 0.71, K1/2 = 66.1 µM without ATP (green). For the control construct zELK-E51Anap without the dihistidine, the quenching data with and without ATP/Mg2+ were merged since ATP/Mg2+ did not produce any significant difference. (C) Inside-out patch-clamp recordings of zELK E51Anap, K729H-E733H channels exhibiting a ATP/Mg2+-dependent slow component of activation typical of VDP in wild-type zELK channels. (D) Representative G-V curves of zELK-E51Anap, K729H-E733H channels exhibiting prepulse-dependent shift in the voltage-dependence of activation typical of VDP in wild-type zELK channels (different patch from panel C). (E) Representative PCF images showing Anap fluorescence decreased when the membrane voltage was stepped from −100 mV to +120 mV for zELK-E51Anap, K729H-E733H channels in the presence of 1 mM Co2+ and ATP/Mg2+ in the bath. (F) Summary data showing the Anap fluorescence decrease by depolarization was abolished without the dihistidines or when the VDP disappeared in the absence of ATP/Mg2+ (n = 5), *p<0.05. The fluorescence was measured using a bandpass filter for Anap emission. (G) Spectral images of L-Anap emission at −100 mV and +120 mV. (H) Emission spectra from the spectral images shown in panel G.