Proton Binding and Conformational Dynamics in KcsA

Abstract

Though KcsA has been the subject of extensive studies, the specific molecular mechanism by which the channel opens in response to proton binding is unknown. We used a multi-pronged approach - electrophysiology, X-ray crystallography, modeling, and NMR - to understand pH-dependent gating in KcsA. We propose that the pH sensor consists of a strong sensor, H25, in addition to one weak sensor, E118. Protonation of these residues drives opening of the channel by shifting the equilibrium from the closed state to the open state. We modeled KcsA pH gating using a modified Monod-Wyman-Changeux (MWC) model with 2 proton binding sites per subunit. In this model, the pKa values for each pH sensor are different in the closed state than in the open state, as required for proton binding to drive opening of the channel. This model choice is reasonable as the conformational changes required to open the channel do produce an alternate environment for H25 and E118, effectively altering their pKa values. We sought to constrain this model by determining the pKa values for these residues directly using solution NMR. using KcsA reconstituted into bicelles, we determined that H25, the strong sensor, responds to pH changes in two distinct conformational states, with an apparent pKa in the range of channel gating. Additional NMR studies targeted at the weak proton sensor, E118, and other bundle-crossing mutants will provide further insight into the coupling of proton binding with channel gating.