Fig. 1: Incorporation of ANAP and IFM Movement during fast inactivation.
A Side view of a Nav channel structure (PDB: 7XVF). IFM motif (in pink) resides in DIII_DIV linker (yellow) and docks into a pocket away from the pore. B Size of ANAP is comparable to tryptophan. With the filter set used in the study (details in Methods), the fluorescence is brighter in hydrophobic environment. C Example ionic current traces of wild type (WT), F1304ANAP, and F1304UAG when no ANAP was supplied. Incorporation of ANAP into IFM motif slowed fast inactivation kinetics. Inset shows the voltage protocol with 5 mV voltage steps. D GV curve of WT and F1304ANAP. No significant difference was observed. N = 4 for WT, N = 5 for F1304ANAP. E h-infinity curve for WT and F1304ANAP, with the latter showing a right-shifted Vhalf (∆Vhalf = ~8.5 mV). Inset shows the voltage protocol with 10 mV voltage steps and a 5 s cycle period. N = 4 for WT, N = 5 for F1304ANAP. F Recovery from fast inactivation at −90mV, measured by comparing the peak current during control and test pulse. F1304ANAP recovered faster than WT, suggesting a less stable inactivated state. N = 4 for WT, N = 5 for F1304ANAP. Inset shows the voltage protocol. G Example family of traces of simultaneously recorded ionic current and fluorescence signal from F1304ANAP. An increase of fluorescence intensity indicates a transition from a relative hydrophilic environment to a more hydrophobic one. Two kinetic components can be distinguished at IFM motif. Left inset shows the voltage protocol with 10 mV voltage steps. Right inset shows the fluorescence signal at 60 mV fitted with only one fast component. H Comparison of steady state fluorescence (FV) curve and h-infinity curve. H-infinity curve is shown as 1-(h-infinity) curve, which closely tracks with the FV curve. N = 5. I Comparison of fluorescence signal and fast inactivation kinetics. The fast fluorescence signal is faster than the fast inactivation. The slow component follows more closely the fast inactivation kinetics. N = 5 for fluorescence signal, N = 4 for ionic current. Data is shown as Mean ± SEM.
