Dynamics of Ion Permeation and Conformational Coupling in – KcsA update, year 1

Summary of Progress

The overall goals of this project are based on a multidisciplinary analysis of the different dynamic transitions that define gating event in K channels. These include relatively slow (ms) helix tilt and hinge-bending motions in the inner bundle gate, very slow (seconds) rearrangements in the selectivity filter (giving rise to C-type inactivation) and very fast (Flicker) transitions also associated with rearrangements at the selectivity filter. Within these wide ranging set of kinetics we plan, in the coming years, to examine the dynamics of ion conduction and the interplay between ion occupancy and selectivity filter conformation.

The types of questions we plan to respond can be approached only because of the robustness and experimental maturity of the system at hand (KcsA). Members of this Project have shown that KcsA represents an ideal system to study fundamental mechanisms of ion permeation and gating, and that these findings are directly applicable to eukaryotic systems. More recently, structure-function experiments led us to identify residues playing a critical role in C-type inactivation, flicker gating and pH sensitivity. Because of these findings, we are in a position to investigate the events linking the different conformations of the channel with the activation and inactivation processes at different time scales. We pursue a concerted approach using semi-synthesis of KcsA, patch clamp, X-ray diffraction and a host of spectroscopic methods (EPR, NMR, and 2D-IR) to develop, with the aid of cutting edge computational methods, an understanding of the conformational changes taking place at the selectivity filter when it conducts ions and when it inactivates.

Research Highlights

Click to find out more about each research highlight (RH).

RH #1. Gating-related Structural Dynamics in the Outer Vestibule of KcsA

RH #2. Synthesis of spin labeled substrates of ABC transporters

RH #3. Structural and dynamic basis of modal gating behavior in K+ channels

Interactions with Cores and Other Sections of the Consortium

The ongoing solution and solid state NMR experiments as well as the upcoming 2D-IR measurements are closely linked to the Chemical Synthesis Core D2 and the Protein Expression and Purification Core D1 for sample preparation, and the Computational Modeling Core D4 for protein dynamics. We expect a continued interaction with the Synthetic Antigen Binder and Crystallography Core D3 to generate novel Fabs to the extracellular regions of KcsA, aiming to generate new, higher resolution structures of KcsA gating intermediates. We will also continue tot exploit our recent success in crystallizing membrane proteins, using conformationally sensitive antibodie frangments. All the computational analysis continue to make extensive use of the spectral simulation tools (2D IR, NMR, ESR) and the transition pathway methodologies (String Method with swarm-of-trajectories) under development by the Computational Core D4.

Team Members
Recent Publications
Summary of Progress
Project Images
Glu71 mutants stabilize individual gating modes in a side-chain specific way.
Glu71 mutants stabilize individual gating modes in a side-chain specific way. (a) Macroscopic responses of wt and various Glu71 mutants elicited by pH jumps from 8.0 to 4.0 using a rapid solution exchanger in the presence of 200 mM KCl and the membrane potential held at +150 mV. The current trace for the E71G mutant is shown at a relative amplitude, compared with the other traces, the inset shows the same trace expanded in the current axis. (b) A plot of Isteady/ Ipeak for various Glu71 mutants (n>5) (c) Single-channel currents were recorded under steady-state conditions at pH 4.0 and +150 mV in 200 mM symmetric K+ solutions. Grey box highlights mutants that are focused in this study. (d) Selectivity versus Na+ estimated from single-channel I-V ramps under bi-ionic conditions. No detectable Na+ currents were seen in any of the mutants. Eapparent is the potential at which K+ currents can last be resolved. Error bars show s.d (n>5)