Probing the Effects of Gating on the Ion Occupancy of the K⁺ Channel Selectivity Filter Using Two-Dimensional Infrared Spectroscopy

Abstract

The interplay between the intracellular gate and the selectivity filter underlies the structural basis for gating in potassium ion channels. Using a combination of protein semisynthesis, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we probe the ion occupancy at the S1 binding site in the constricted state of the selectivity filter of the KcsA channel when the intracellular gate is open and closed. The 2D IR spectra resolve two features, whose relative intensities depend on the state of the intracellular gate. By matching the experiment to calculated 2D IR spectra of structures predicted by MD simulations, we identify the two features as corresponding to states with S1 occupied or unoccupied by K+. We learn that S1 is >70% occupied when the intracellular gate is closed and <15% occupied when the gate is open. Comparison of MD trajectories show that opening of the intracellular gate causes a structural change in the selectivity filter, which leads to a change in the ion occupancy. This work reveals the complexity of the conformational landscape of the K+ channel selectivity filter and its dependence on the state of the intracellular gate.

Structure, function, and ion-binding properties of a K + channel stabilized in the 2,4-ion–bound configuration

“…This overzealous scientific attention is based on its' role in catalyzing an unparalleled rapid and highly ion-selective transport. The molecular mechanism underlying this unique function has been the subject of functional, structural, and computational studies and yet remains a controversial topic of research (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16).…”

“…Despite substantial experimental evidence and computational studies supporting these 2 different mechanistic proposals, a clear consensus is yet to be reached regarding the universality of a model for ion conduction in K + channels. As a result, this topic has reemerged as highly controversial (7)(8)(9)19).…”

Modulation of Function, Structure and Clustering of K+ Channels by Lipids: Lessons Learnt from KcsA

“…Conversely, the complexity observed in permeation features (i.e., low open probability for K + and very diminished inactivation in the presence of Rb + or Cs + ) cannot be explained just in terms of binding at equilibrium and likely relates to reported differences in the occupancy of the S2 and S3 sites by the permeant cations and/or kinetics contributions. In addition, some recent experiments performed by 2D-IR spectroscopy found that the occupation of the S1 site diminishes upon channel opening [63].…”

Two-dimensional infrared (2D IR) spectroscopy for elucidating ion occupancies in the selectivity filter of ion channels1

“…We not only provide new experimental evidence that support the knock-on model of ion conduction but also present new spectroscopic signatures for a Val76-flipped state which has only been observed in simulations. Our second study focuses on the coupling between the selectivity filter and the helix bundle gate, inferred through changes in the ion occupancy of the filter under different experimental conditions [34]. Using the same techniques, we probed the ion occupancies in the selectivity filter of KcsA in the closed-constricted and the open-constricted states.…”

“…While the details of the MD simulations are provided elsewhere [33,34]. Briefly, for our initial studies, we performed MD simulations using Drude oscillator derived polarizable force fields starting from previously described equilibrated structures [38].…”