Ion-Selectivity Filter Interactions in KCSA from 1D 87Rb+ NMR

Abstract

In K+ channels, permeation/selectivity and gating at the selectivity filter are all intimately influenced by the energetics and dynamics of ion-protein interactions. Specific contacts between carbonyl groups and the permeant ion define its high selectivity although the high transport rate suggests that ion-binding interactions must have rather small energies.Binding constants for alkali cations have been reported for several K+ channels. However, it is difficult to determine equilibrium constants for weakly bound systems, and there are large discrepancies in these values. One-dimensional NMR has the unique ability to directly report binding interactions between ions and their binding pocket, as the case for studies of potassium coordination in gramicidin. Here we carried out 1D 87Rb+NMR (a spin 3/2 ion with adequate natural abundance) in bicelle-reconstituted full-length KcsA to study ion interactions at the K+ selectivity filter.Using chemical shift and linewidth analysis, we developed a simple two-state model that adequately reproduces the data at higher ion concentration (>10 mM). However, predicted values deviate from observed data at lower millimolar concentrations, suggesting a more complex mechanism of interaction. Two factors are considered: the presence of two binding sites as well as filter conformational changes in ion-depleted complexes. In addition, a Kon rate of approximately 108 M-1 s-1 was estimated with our model, in overall agreement with the fast rate of permeation in ions channels. Our ability to measure the relationship between ion concentration and the chemical shift and linewidth experimentally and recapitulate the findings with a simple two state model suggests that this technique could be valuable in studying the dynamics of a variety of permeant ions and may improve our understanding of interactions between ion and the K+ channel selectivity filter.