Xenbase Image ID: 170225

Figure 5. Measuring the ATP/Mg2+-dependent and voltage-dependent change in the distance between the eag domain and CNBHD of zELK channels using tmFRET.(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.DOI: http://dx.doi.org/10.7554/eLife.26355.009Figure 5—figure supplement 1. tmFRET between L-Anap and transition metal ions.(A) Spectra of L-Anap excitation and emission and absorbance (measured as extinction coefficient) of cobalt and copper coordinated by a dihistidine pair. (B) Förster distance of the L-Anap/Co2+-dihistidine FRET pair (labeled with the dashed line).DOI: http://dx.doi.org/10.7554/eLife.26355.010 Image published in: Dai G and Zagotta WN (2017) © 2017, Dai et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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