ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of the Selectivity Filter Interacting with K(+) and Na(+) in the Open and Collapsed Conformations of the KcsA Potassium Channel.

The potassium channel is highly selective for K(+) over Na(+), and the selectivity filter binds multiple dehydrated K(+) ions upon permeation. Here, we applied attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to extract ion-binding-induced signals of the KcsA potassium channel at neutral pH. Shifts in the peak of the amide-I signal towards lower vibrational frequencies were observed as K(+) was replaced with Na(+). These ion species-specific shifts deduced the selectivity filter as the source of the signal, which was supported by the spectra of a mutant for the selectivity filter (Y78F). The difference FTIR spectra between the solution containing various concentrations of K(+) and that containing pure Na(+) demonstrated two types of peak shifts of the amide-I vibration in response to the K(+) concentration. These signals represent the binding of K(+) ions to the different sites in the selectivity filter with different dissociation constants (KD = 9 or 18 mM).