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

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.

Automated summary

The potassium ion configurations in the selectivity filter of the KcsA ion channel protein were investigated using 2D IR spectroscopy. The study found that in the closed conductive state of the KcsA channel, K+ ions do not occupy adjacent binding sites. The experimental data supported the soft-knock ion configurations rather than the hard-knock ion configurations.