Probing Ion Configurations in the KcsA Selectivity Filter with Single-Isotope Labels and 2D IR Spectroscopy
Ryan, MJ (Ryan, Matthew J.); Gao, LJ (Gao, Lujia); Valiyaveetil, FI (Valiyaveetil, Francis I.); Zanni, MT (Zanni, Martin T.); Kananenka, AA (Kananenka, Alexei A.)
Journal of the American Chemical Society, 2023, Volume 145, 18529-18537.
The potassium ion(K+) configurations of the selectivityfilter of the KcsA ion channel protein are investigated with two-dimensionalinfrared (2D IR) spectroscopy of amide I vibrations. Single C-13-O-18 isotope labels are used, for the first time,to selectively probe the S1/S2 or S2/S3 binding sites in the selectivityfilter. These binding sites have the largest differences in ion occupancyin two competing K+ transport mechanisms: soft-knock andhard-knock. According to the former, water molecules alternate betweenK(+) ions in the selectivity filter while the latter assumesthat K+ ions occupy the adjacent sites. Molecular dynamicssimulations and computational spectroscopy are employed to interpretexperimental 2D IR spectra. We find that in the closed conductivestate of the KcsA channel, K+ ions do not occupy adjacentbinding sites. The experimental data is consistent with simulated2D IR spectra of soft-knock ion configurations. In contrast, the simulatedspectra for the hard-knock ion configurations do not reproduce theexperimental results. 2D IR spectra of the hard-knock mechanism havelower frequencies, homogeneous 2D lineshapes, and multiple peaks.In contrast, ion configurations of the soft-knock model produce 2DIR spectra with a single peak at a higher frequency and inhomogeneouslineshape. We conclude that under equilibrium conditions, in the absenceof transmembrane voltage, both water and K+ ions occupythe selectivity filter of the KcsA channel in the closed conductivestate. The ion configuration is central to the mechanism of ion transportthrough potassium channels. |
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