Mutant of a Light-Driven Sodium Ion Pump Can Transport Cesium Ions

<i>Krokinobacter eikastus</i> rhodopsin 2 (KR2) is a light-driven Na⁺ pump found in marine bacterium. KR2 pumps Li⁺ and Na⁺, but it becomes an H⁺ pump in the presence of K⁺, Rb⁺, and Cs⁺. Site-directed mutagenesis of the cytoplasmic surface successfully converted KR2 into a light-driven K⁺ pump, suggesting that ion selectivity is determined at the cytoplasmic surface. Here we extended this research and successfully created a light-driven Cs⁺ pump. KR2 N61L/G263F pumps Cs⁺ as well as other monovalent cations in the presence of a protonophore. Ion-transport activities correlated with the additive volume of the residues at 61 and 263. The result suggests that an ion-selectivity filter is affected by these two residues and functions by strict exclusion of K⁺ and larger cations in the wild type (N61/G263). In contrast, introduction of large residues possibly destroys local structures of the ion-selectivity filter, leading to the permeation of K⁺ (P61/W263) and Cs⁺ (L61/F263)

Simulation systems used in this research.

<p>The simulations performed in this research. In this table, “p” and “Δp” refer to protonated and deprotonated glutamate, respectively.</p><p>Simulation systems used in this research.</p

Effects of deprotonation of both Glu122 and Glu129 in the ground state.

<p>Structural comparison of the intracellular constrictions between the ATR-E122p-E129p and ATR-E122Δp-E129Δp simulations. (A) Overall structures of snapshots from the last frame of both simulations. Key residues are highlighted in orange and magenta. (B) Magnified view of the intracellular and central constrictions (left and right panels, respectively). Double arrows indicate the possible motions of Glu121-Arg307 (red), Glu122-His173 (cyan), Glu122-Arg307 (green) and Glu129-Asn297 (magenta). (C-F) Distances between (C) Glu121-Arg307, (D) Glu122-His173, (E) Glu122-Arg307, and (F) Glu129-Asn297. (G, H) Distribution of water molecules in the (G) ATR-E122p-E129p and (H) ATR-E122Δp-E129Δp simulations. The distribution maps are contoured at the probability density of 0.0015 molecules Å^-3 ns^-1. The time-averaged structure of the protein over 150 ns is shown.</p

Effects of the deprotonation of either Glu122 or Glu129 in the ground state.

<p>(A, B, C) Distances between (A) Glu129-Asn297, (B) Glu122-His273 and (C) Glu122-Arg307 in the ATR-E122p-E129Δp and ATR-E122Δp-E129p simulations. (D, E) Distributions of water molecules in the ATR-E122p-E129Δp and ATR-E122Δp-E129p simulations. The distribution map is contoured at the probability density of 0.0015 molecules Å^-3 ns^-1. The time-averaged structure of the protein over 150 ns is shown.</p

Electrophysiological analysis for C1C2 variants.

<p>(A) The peak amplitudes of the photocurrents, normalized by the cell’s input capacitance. (B) Conforcal images of representative HEK293 cells expressing the C1C2 WT and its mutants. Scale bar represents 30 μm. (C) The expression level of each C1C2 variant measured by the membrane/cytosol ratio of GFP fluorescence. (D-G) The current-voltage (<i>I-V</i>) relation curves for each mutant. (H, I) The kinetic parameters for each mutant, (H) opening rates (τ_on) and (I) closing rates (τ_off). The error bars represent s.e.m. of 3 experiments (n = 5–17 cells). * p < 0.05.</p

The two constrictions observed in the crystal structure of C1C2.

<p>The two constrictions in the crystal structure of C1C2 (PDB ID 3UG9) and the electrophysiological analysis of the constrictions. (A) Overall structure of C1C2, viewed parallel to the membrane with the three key regions highlighted (magenta, blue, and red). The dashed area represents the putative ion-conducting pathway. (B) Magnified views of the highlighted regions in (A). Black dashed lines are hydrogen bonds, and orange dashed circles represent the putative conducting pathway.</p

Correlation analysis for the 13-<i>cis</i>R-122Δp-129p simulation.

<p>(A) The matrix of correlation coefficients for the pairs of Cα atoms. (B) Mapping of the correlation coefficients to the structure. The black dashed circle represents the pair of Cα atoms with a correlation coefficient greater than 0.7. The red dashed circle represents the pair of Cα atoms in TM2 and TM7 that has a negative correlation coefficient.</p

The conformational change in Trp262 upon retinal isomerization.

<p>(A) Structural comparison between the snapshots from the ATR-bound (grey) and 13-<i>cis</i>R-bound (green) simulations. (B) Magnified view of retinal and Trp262, from the orange-highlighted region in the left panel. Double arrows indicate the possible motions of Trp262. (C) The RMSD values of the Trp262 atoms, relative to those of the crystal structure. (D) The peak amplitudes of the photocurrents, normalized by the cell’s input capacitance. (E) Conforcal images of representative HEK293 cells expressing the C1C2 WT and W262A mutants. Scale bar represents 30 μm. (F) The expression level of W262A mutant measured by the membrane/cytosol ratio of GFP fluorescence. The error bars represent s.e.m. of 3 experiments (n = 5–17 cells). * <i>p</i> < 0.05.</p

The movements of TM helices upon retinal isomerization.

<p>(A) Structural comparison between the snapshots from the ATR-bound (grey) and 13-<i>cis</i>R-bound (green) simulations. (B) Magnified cytoplasmic view of the red-highlighted region in the left panel. (C-E) The RMSD values of (C) TM6, (D) TM7 and (E) TM2, compared between the ATR-bound and 13-<i>cis</i>R-bound forms. (F) Distance between Glu121-Arg07 in the intracellular constriction.</p