Fig. 5: Pore movement as the last conformational step in fast inactivation.

A To track the pore movement during fast inactivation, ANAP was incorporated at Q1293 position at the bottom of DIII S6 (green). B Example of ionic current and fluorescence signal recorded from Q1293ANAP. Inset shows overlapped comparison of ionic current and fluorescence signal at 60 mV. There is a ~ 1 ms delay to the onset of fluorescence signal, suggesting a sequential movement for fast inactivation. C, D GV curve and h-infinity curve for Q1293ANAP. No drastic difference was observed. N = 5 for Q1293ANAP. E Comparison of fluorescence kinetics and fast inactivation kinetics. Fluorescence kinetics follows closely the fast inactivation development, suggesting the conformational change observed at this position reports mostly pore movement associated with fast inactivation. N = 10. F) Example of ionic current and fluorescence signal from L1319ANAP_F1291A. G Comparison of fluorescence kinetics between L1319ANAP and L1319ANAP_F1291A. No significant difference in kinetics is seen in the DIII_DIV linker movement after abolishing the interaction between IFM and F1291, favoring a sequential model for fast inactivation. N = 7 for L1319ANAP_F1291A and N = 8 for L1319ANAP. Data is shown as Mean ± SEM.