Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy

Abstract

Potassium channels are responsible for the selective permeation of K⁺ ions across cell membranes. K⁺ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K⁺ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (¹³C¹⁸O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K⁺ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

Figure 1. Overview of Ion Permeation Mechanisms of KcsA

(A) X-ray crystal structure of KcsA (PDB ID 1K4C) with two of the four protein subunits shown. (B) The selectivity filter of KcsA is lined with the carbonyl groups (oxygen in red) of four amino acids (Thr75, Val76, Gly77, and Tyr78). Amino acids that were ¹³C¹⁸O isotope labeled for the experiment are shown bolded (Val76, Gly77, and Gly79). The binding sites (S1 to S4) span the selectivity filter. (C) Knock-on mechanism for potassium ion permeation through the channel. In this mechanism, K⁺ (purple), alternate with water molecules (red) and collectively move through the filter when a new K⁺ enters the filter. (D) The two most prominent ion-binding configurations for the knock-on mechanism used to simulate the 2D IR spectra: (left) [S1,W,S3,W] and (right) [W,S2,W,S4]. (E) The hard-knock model for ion conduction. When a K⁺ ion enters the S4 binding site, strong electrostatic repulsions simultaneously translocate two ions upward. No water is involved in this mechanism. (F) The two ion configurations used to simulate the 2D IR spectra: (left) [0,S2,S3,0] and (right) [S1,S2,0,S4]. Simulations for 97% of the ion configurations proposed by the hard-knock mechanism are shown in the SM, Fig. S7.

Figure 2. Experimental 2D IR spectra of unlabeled and ¹³C¹⁸O-labeled KcsA at high potassium concentrations

(A) Pulse sequence for 2D IR spectrum with illustrated vibrational coherences (blue). Fourier transform of the coherence during t_probe to give the ω_probe axis while the time between the pump pulses, t_pump, is computationally Fourier transformed to give the ω_pump axis. (B) 2D IR spectrum of unlabeled KcsA showing the absorption of side chains in the region where the isotope labeled features are expected to appear. (C) 2D IR spectrum of labeled KcsA shows a 50% increase in intensity in this region, consistent with absorption by the ¹³C¹⁸O labeled residues. (D) Subtraction of the unlabeled spectrum from the labeled spectrum yields two pairs of peaks. The bleaches (blue) appear at (ω_pump,ω_probe )= (1603 cm⁻¹, 1609 cm⁻¹) and (1580 cm⁻¹, 1582 cm⁻¹), respectively. (E) For every unique combination of difference spectra, the intensities of the 3x3 pixel array centered at the pixels for (ω_pump, ω_probe) = (1603 cm⁻¹, 1609 cm⁻¹) (peak1) and (1580 cm⁻¹, 1582 cm⁻¹) (peak 2) were integrated, respectively. The slope of the fit yielded an intensity ratio of 1.25 ± 0.02 with an R-square of 0.95.

Figure 3. Calculated 2D IR spectra to test proposed ion configurations of the KcsA selectivity filter

The calculated 2D IR spectra for the (A) [W,S2,W,S4], (B) [S1,W,S3,W], (C) [S1,W,S3,W] with a single Val76 flip, (D) [0,S2,S3,0], and (E) [S1,S2,0,S4] K⁺ binding configurations of the labeled KcsA filter from MD simulations using a K⁺ charge profile described in (35). In (A), we have the spectrum for the [W,S2,W,S4] ion binding configuration in which all of the Val76 residues point into the filter. In this case, we observe peaks at (ω_pump, ω_probe) = (1608 cm⁻¹, 1612 cm⁻¹). (B) For the [S1,W,S3,W] configuration, we see a similar spectrum with (ω_pump, ω_probe) = (1600 cm⁻¹, 1604 cm⁻¹). (C) For the [S1,W,S3,W] ion configuration of the Val76-flipped state, we get a pair of inhomogeneous peaks at (ω_pump, ω_probe) = (1583 cm⁻¹, 1587 cm⁻¹). Fig. 3(D) and (E) are the calculated 2D IR spectra of the [0,S2,S3,0] and [S1,S2,0,S4] binding configurations, respectively. In (E), we observe a single homogenous peak at (ω_pump, ω_probe) = (1587 cm⁻¹, 1591 cm⁻¹), which is similar to the homogeneous peak we observe in (F) at (wpump, Wpwbe) (1585 ern·′, 1589 ern⁻¹. The spectra have been normalized for visualization purposes.

Figure 4. Comparison of 2D IR experimental spectrum to the simulated spectra of the knock-on models (with and without water) and the hard-knock model

(A) Experimental difference spectrum shown in Fig. 2C. (B) Simulated 2D IR spectrum for the knock-on model with water generated by a weighted average of the [W,S2,W,S4], [S1,W,S3,W], and Val76 flipped [S1,W,S3,W] ion configurations in a ratio of 0.3:0.3:0.4, respectively. The simulated spectrum agrees well with peak positions, 2D lineshapes, and intensity ratio. (C) Simulated 2D IR spectrum for the hard-knock model from a weighted average of [0,S2,S3,0] and [S1,S2,0,S4] along with other hard-knock states (see SM). (D) Simulated 2D IR spectrum of the knock-on model with water removed with the same ratios of states used to match experiment (Fig. 4B). No linear combination of these states without water (see Fig. S14) reproduces the experimental spectrum. Therefore, water must be present within the filter. (E–H) Zoom-ins of the high frequency peak of each spectra. The nodal line slopes are illustrated by red lines with values of the inverse slope (IS).