Quantitative Analysis of the Water Occupancy around the Selectivity Filter of a K⁺ Channel in Different Gating Modes

Abstract

Recovery in K+ channels, i.e., the transition from the inactivated non-conductive selectivity filter conformation towards the conductive conformation, occurs on a timescale of the order of seconds, which is astonishingly long, given that the structural differences among the filter conformations are faint (< 1 Å). Computational studies and electrophysiological measurements suggested that buried water molecules bound behind the selectivity filter are at the origin of the slowness of recovery in K+ channels. Using a combination of solid-state NMR spectroscopy (ssNMR) and long molecular dynamics simulations, we sketch a high-resolution map of the spatial and temporal distribution of water behind the selectivity filter of a membrane-embedded K+ channel in two different gating modes. Our study demonstrates that buried water molecules with long residence times are spread all along the rear of the inactivated filter, which explains the recovery kinetics. In contrast, the same region of the structure appears to be dewetted when the selectivity filter is in the conductive state. Using proton-detected ssNMR on fully protonated channels, we demonstrate the presence of a pathway that allows for the interchange of buried and bulk water, as required for a functional influence of buried water on recovery and slow inactivation. Furthermore, we provide direct experimental evidence for the presence of additional ordered water molecules that surround the filter and that are modulated by the channel’s gating mode.

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

“…Figure 2 A shows a 2D NH spectrum of iFD/[ 13 C, 15 N]‐labeled KcsA, reconstituted in E. coli lipids and protonated buffers, acquired at 60 kHz MAS and 800 MHz. This spectrum, which shows the H N signals of the entire channel, is already of superior resolution compared to our earlier results with fully protonated KcsA,3c and readily allows identifying previously assigned water‐exposed signals (red, Figure 2 A) 2f. To exclusively select the TM part, we incubated iFD‐labeled KcsA in D 2 O and acquired a 2D NH spectrum, which featured a stark enhancement in spectral quality with well‐resolved signals as narrow as 0.13 ppm (Figures 2 B, C).…”

“…It is a particularly severe problem for the study of membrane proteins, whose transmembrane (TM) parts are critical for their function and do not undergo exchange in protonated buffers 2c, 3c, 5. This prompted us to develop a two‐step approach that is generally applicable in vitro and in situ, works already at moderate MAS frequencies of 60 kHz, and provides well‐resolved 1 H‐detected NMR spectra of water‐inaccessible protein regions.…”

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

“…The transition rates between the two principal gating states are described by conformational changes in the pore region (including the inactivation gate and turret). In detail, however, the degree of opening and closing of both gates is determined by the combined influence of protein sequence, lipid environment, and small molecules such as water molecules (56) or cations such as potassium and sodium. Together, they control the delicate conformational coupling of the inactivation and activation gates in membranes in each state of the potassium channel gating cycle.…”

On the role of water density fluctuations in the inhibition of a proton channel

“…The latter were also observed in biological systems as a consequence of minor changes of the hydrophobic environment, as in the case of the melittin tetramer (100). Interestingly, wet/dry transitions in protein pockets have been shown to be of crucial functional relevance in K + channels, where they drive the transition between the conductive and inactivated states of the selectivity filter (101,102).…”