Probing Ion Configurations in the KcsA Selectivity Filter with Single-Isotope Labels and 2D IR Spectroscopy

[Image: see text] The potassium ion (K(+)) configurations of the selectivity filter of the KcsA ion channel protein are investigated with two-dimensional infrared (2D IR) spectroscopy of amide I vibrations. Single (13)C–(18)O isotope labels are used, for the first time, to selectively probe the S1/S2 or S2/S3 binding sites in the selectivity filter. These binding sites have the largest differences in ion occupancy in two competing K(+) transport mechanisms: soft-knock and hard-knock. According to the former, water molecules alternate between K(+) ions in the selectivity filter while the latter assumes that K(+) ions occupy the adjacent sites. Molecular dynamics simulations and computational spectroscopy are employed to interpret experimental 2D IR spectra. We find that in the closed conductive state of the KcsA channel, K(+) ions do not occupy adjacent binding sites. The experimental data is consistent with simulated 2D IR spectra of soft-knock ion configurations. In contrast, the simulated spectra for the hard-knock ion configurations do not reproduce the experimental results. 2D IR spectra of the hard-knock mechanism have lower frequencies, homogeneous 2D lineshapes, and multiple peaks. In contrast, ion configurations of the soft-knock model produce 2D IR spectra with a single peak at a higher frequency and inhomogeneous lineshape. We conclude that under equilibrium conditions, in the absence of transmembrane voltage, both water and K(+) ions occupy the selectivity filter of the KcsA channel in the closed conductive state. The ion configuration is central to the mechanism of ion transport through potassium channels.