Figure 2: Measurement of H4 di-histidine using transition-metal ion FRET is similar to the NMR structure and does not affect function.
From: Perturbation of sodium channel structure by an inherited Long QT Syndrome mutation
(a) Model of the NaV1.5 CT EF-hand domain from the solution NMR structure by Chagot et al. (2009). Introduced histidines on the fourth helix (H4) are modelled in magenta. The tryptophan on the first helix (H1) used as the fluorescence donor is modelled in orange. (b) Representative tryptophan emission dose–response spectra of NaV1.5 CT 1943 (WT) with increasing concentrations of Ni2+. (c) Representative tryptophan emission dose–response spectra of NaV1.5 CT H4 di-HIS (F1855H/K1859H). (d) Fluorescence quenching of NaV1.5 CT WT (black) and H4 di-HIS (magenta) in the presence of Ni2+. Curves are averaged-normalized fits with a modified Hill equation (see methods). Points are averaged-normalized individual data points±s.d. (e) WT (grey) and H4-diHis (black) NaV1.5 traces shown at low gain and high gain to show absence of persistent current in both. Vertical scale bars represent 10% peak current for low-gain and 2% peak current for high-gain insets. Horizontal scale bars represent 25 ms for low-gain and 32 ms for high-gain insets (f) Introduction of H4 di-histidine mutations does not increase persistent Na current. Scale bars represent sustained current (%) to peak current at 150 ms for WT (n=7) and H4 di-HIS (n=6). Scale bars are mean±s.d.
