Dynamics of Conformational Transitions during KcsA Gating from EPR and Solution NMR Experiments

Abstract

Despite its apparent simplicity, the potassium channel KcsA possesses a relatively complex gating cycle. Previous studies show conformational changes at the inner gate with an increase in distance from the central axis of symmetry when activated. These changes, coupled to rearrangements at the selectivity filter, are hypothesized to underlie activation, inactivation and modal gating. Defining the dynamic properties of the conformational wave linking the inner gate and selectivity filter is a critical step in understanding the mechanism of gating and permeation in K+ channels.Here, we have used CW and DEER Electron Paramagnetic Resonance to measure the opening of the inner gate and NMR to investigate the dynamics of the backbone of KcsA in a pH dependent manner. KcsA G116C was spin-labeled and reconstituted into liposomes or DDM micelles for EPR studies. NMR studies used wild type KcsA Δ125 in micelles (from pH7 to pH3 in 0.5 unit increments) with 2H/15N labeling and a TROSY pulse sequence. Pilot experiments investigating dynamics used a CPMG pulse sequence at pH7 and pH3.The extent of conformational dynamics from the inner gate ranged from 10.0 A (closed, pH7) to 23.7 A (open, pH3) with pH activation. Distance changes were abrupt near the pKa of activation (pH 3.9). Importantly, an observed increase in the width of the population distribution, and hence dynamics, was also observed at this point. Preliminary NMR experiments confirm that this region possesses distinct chemical shifts from a closed versus an open, inactivated state.Ongoing experiments target the process of backbone dynamics at different regions of KcsA. New pulse sequence schemes and kinetic gating mutants of KcsA are being studied to isolate the multiple dynamics components that define KcsA modal gating.