Dynamics of the KcsA Selectivity Filter Probed using Intrinsic Tyrosine Fluorescence

KcsA is activated by intracellular protons through a large conformational change in the inner helical-bundle gate. The selectivity filter and surrounding structures play a crucial role in ion conduction as an inactivation gate. To monitor the conformational dynamics of the selectivity filter under various experimental conditions we engineered a KcsA construct devoid of tryptophan and tyrosine residues with the exception of residue Y78, locatd in the middle of the selectivity filter. Fluorescence lifetimes indicate that the microenvironment polarity of Y78-KcsA decreases upon gating, irrespective of the type of ion (K+, Rb+, NH4+, Cs+) present in the selectivity filter. Further, time-resolved anisotropy measurements indicate that the dynamics associated with the segmental mobility of the selectivity filter is significantly restricted, and this gating-driven motional restriction is more pronounced in High K+ and Rb+ conditions. Red Edge Excitation Shift (REES) measurements of Y78-KcsA show slow water relaxation dynamics under conditions that stabilize conductive and non-conductive filter conformations, suggesting the presence of bound/restricted water molecules in both filter conformations. Interestingly, opening the lower gate appears to further restrict the water molecules in the vicinity of the selectivity filter. Overall, our results show that the conformational dynamics associated with the selectivity filters of collapsed (Low K+) and open-inactivated KcsA are significantly different despite showing essentially identical conformations as derived from crystal structures. This highlights the importance of hydration dynamics in gating of K+ channels.