Ion-protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy

Abstract

An understanding of ion-protein interactions is key to a better understanding of the molecular mechanisms of proteins, such as enzymes, ion channels, and ion pumps. A potassium ion channel, KcsA, has been extensively studied in terms of ion selectivity. Alkali metal cations in the selectivity filter were visualized by X-ray crystallography. Infrared spectroscopy has an intrinsically higher structural sensitivity due to frequency changes in molecular vibrations interacting with different ions. In this review article, I attempt to summarize ion-exchange-induced differences in Fourier transform infrared spectroscopy, as applied to KcsA, to explain how this method can be utilized to study ion-protein interactions in the KcsA selectivity filter. A band at 1680 cm^-1 in the amide I region would be a marker band for the ion occupancy of K⁺, Rb⁺, and Cs⁺ in the filter. The band at 1627 cm^-1 observed in both Na⁺ and Li⁺ conditions suggests that the selectivity filter similarly interacts with these ions. In addition to the structural information, the results show that the titration of K⁺ ions provides quantitative information on the ion affinity of the selectivity filter.

Keywords: Infrared spectroscopy; Ion channel; Ion–protein interactions; Membrane proteins.

Fig. 1

X-ray crystal structure of KcsA. a Top (left) and side (right) view of the entire KcsA, a K⁺ ion channel, transmembrane region (PDB: 1K4C). Four monomers are colored in red, yellow, blue, and orange, respectively. The green spheres represent K⁺ ions in the pore. b–d The selectivity filter region (TVGYG) was excised from two monomers with K⁺ ions (b high concentration, c low concentration) or Na⁺ ions (d). PDB codes of the structural models in b, c, and d: 1K4C, 1K4D, and 2ITC, respectively. e Schematic illustrations of ionic radii of alkali metal cations (Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺) scaled against a water molecule [the values are ångströms (Å) obtained from the Pauling estimation; Hille 2001] and van der Waals radii for H and O

Fig. 2

Difference infrared spectra of KcsA in the amide I region. a Ion-exchange-induced spectra differences (K⁺ − Na⁺) with different concentrations of K⁺. b The difference spectra were scaled for similar intensities at 1659, 1650, 1639, and 1627 cm⁻¹. The positive sides measured different K⁺ and Na⁺ ratios at a total concentration of 200 mM (K⁺ = X mM, Na⁺ = 200 − X mM, where X = 3, 10, 20, 50, 100, and 200 mM). The negative sides always measured a 200 mM Na⁺ concentration

Fig. 3

X-ray crystal structures in the selectivity filter region accommodating Li⁺ (a), Rb⁺ (b), and Cs⁺ ions. The position of Li⁺, shown as a sky-blue circle, was hypothesized according to the results of an molecular dynamics simulation. The others (Rb⁺, purple spheres; Cs⁺, yellow spheres) were modeled based on electron densities. PDB codes of the structure models in a, b, and c are 3GB7, 1R3I, and 1R3L, respectively

Fig. 4

Difference infrared spectra of KcsA in the amide I region. a Ion-exchange-induced spectra differences when K⁺ is replaced with Li⁺ (sky-blue line), Rb⁺ (purple line), or Cs⁺ (yellow line). b Ion-exchange-induced spectra differences when Na⁺ is replaced with Li⁺, Rb⁺, or Cs⁺ (same colors as in a). The K⁺ − Na⁺ spectrum difference (gray line) is superimposed on each spectrum difference for comparison. The gray dotted lines are spectra differences collected without exchanging the buffer solutions