Bridge 4: Dynamics of Ion Permeation and Conformational Coupling in K+channels

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Protein-protein and protein-H2O hydrogen bonds have been implicated in defining selectivity filter dynamics and overall function. In Phase II of this consortium, we will continue to study these events by combining spectroscopic, crystallographic, electrophysiological and computational methods to probe the dynamics of the different gating modes in K+ selective filters. In addition to our basic experimental model (KcsA) we have now have added a second system with remarkable selectivity filter plasticity in regards to overall conformation. The NaK-hERG construct undergoes dramatic structural rearrangements in response to both changes in permeant ions and as a result of clinically relevant mutations (associated with the long QT syndrome). Because of these findings, we are in a position to analyze, at atomic level, a wider universe of conformational dynamics at the selectivity filter through multiple time scales. This unique structural dynamics dataset will allow us to pursue detailed computational analyses to provide a definitive description of the energy landscape that connects each static structure with its conformational pathway and associated functional consequences. We propose a concerted approach using cell free and semi-synthesis of KcsA and NaK-hERG, patch clamp, X- ray diffraction and state of the art spectroscopic methods (NMR and ssNMR) to develop, with the aid of cutting edge computational methods, an understanding of the conformational changes taking place at the selectivity filter when it inactivates and during ion conduction. These overall goals will be carried out through the following specific aims: 1. Determine the molecular motions, overall energetics and role of ion interactions associated with the conformational flexibility of K+ selective filter in KcsA and a NaK-hERG chimera. 2. Establish the role of selectivity filter structural water dynamics on the kinetics of C-type inactivation in KcsA and the energetic rules that connect all key gating intermediates using a combination of experimental and computational approaches. The types of questions we plan to address can be approached only because of the robustness and experimental maturity of the systems at hand (KcsA and NaK-hERG). Estimation of the energy landscape for the universe of conformations together with high bandwidth electrophysiology will allow us to establish the general rules that describe a wide range of motion, dynamics and gating modes associated with the K+ selectivity filter. This data is expected to provide a firm understanding of selectivity filter gating mechanisms in the two model systems and is expected to also apply to other members of the K+ channel superfamily.