Gating-Related Conformational Changes in the Outer Vestibule of KcsA: a Fluorescence and Pulsed-Epr Analysis

In K+ channels, the selectivity filter and surrounding structures play a crucial role in inactivation gating and flicker. KcsA is a pH-gated K+ channel and its gating is modulated by transmembrane voltage. In this work, we monitored the gating-related structural dynamics in the outer vestibule of KcsA using site-directed NBD fluorescence and pulsed-EPR analysis. Fluorescence polarization results show that in KcsA, the dynamics of the outer vestibule is substantially different when comparing inactivating (wild type) and non-inactivating (E71A) forms of the channel. In addition, the rate of solvent relaxation (dynamics of hydration) is found to be faster in non-inactivating form of KcsA upon gating as determined by red edge excitation shift (REES) analysis. This increased rate of solvent relaxation correlates well with the increased rotational mobility of the outer vestibule residues in the open, non-inactivating state (E71A at pH 4). To gain further insight on the dynamic properties of these conformational fluctuations in the outer vestibule of KcsA during gating, four pulse Double-Electron-Electron Resonance (DEER) EPR spectroscopy is being currently used. This approach allows for the determination of inter-subunit distances between 20-60 A, directly informing on the overall distance distribution. We have used tandem dimer constructs of spin-labeled KcsA for the residues corresponding to the outer vestibule of KcsA to determine average distances and distance distributions at low pH, under conditions that stabilize the inactivated (wild-type filter) and the non-inactivating (E71A) states. The results will be discussed in terms of the conformational transitions in the outer vestibule during activation and inactivation gating.