Structural Titration of Slo2.2, a Na+-Dependent K+ Channel

Abstract

The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na⁺-activated K⁺ channel, as a function of the Na⁺ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na⁺ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na⁺-dependent manner, without evidence of Na⁺-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.

Keywords: K(+) channel; Slo2 channel; channel gating mechanism; cryo-electron microscopy; high conductance K(+) channel; ion channel gating; ligand-gated ion channel; molecular structure.

Figure 1. Heterogeneity of Slo2.2 particles in 20 – 160 mM NaCl

(A) Ten 3D class averages of Slo2.2 vitrified in the presence of 20 mM, 40 mM, 80 mM and 160 mM Na⁺. The nine classes that closely resemble closed, Na⁺-free Slo2.2 are colored red and the open class is colored blue. (B) Fraction of particles in each class is shown for the four Na⁺ concentrations. The sum of fractions of all ten classes for each Na⁺ concentration is 1.0.

Figure 2. Cryo-EM structure of Slo2.2 in open and closed conformations

(A, B) Cryo-EM density maps of open (A) and closed (B) Slo2.2 low-pass filtered to 3.8 Å resolution and 4.3 Å resolution, respectively, calculated from images of Slo2.2 vitrified in the presence of 300 mM Na⁺. (C, D) Ribbon diagrams of open (C) and closed (D) Slo2.2 colored by domain: S1–S4 domains green, pore domains yellow, RCK1 domains blue and RCK2 domains red. Grey lines indicate approximate borders of the membrane. See also Figures S1, S2, S3 and S7.

Figure 3. Activation of Slo2.2 by intracellular Na⁺

(A) Superposition of open and closed Slo2.2 channels aligned by the RCK2 domains of all four subunits. Front and rear subunits, S1–S4 domains and S5 are removed for clarity. The pore and RCK1 N-lobes are colored blue and red in the open and closed structures, respectively. Dashed lines indicate residues absent from the closed structure. (B) Superposition of open (blue) and closed (red) pore domains aligned by the pore helices and selectivity filters viewed from within the plane of the membrane (left) and from the cytoplasm (right). Spheres represent the Cα position of Met-333. (C) Plot of pore diameter (between van der Waals surfaces) for open (blue) and closed (red) Slo2.2. The narrowest part of the pore in closed Slo2.2 below the selectivity filter is located at Met-333 (6.2 Å). (D,E) Representative currents at 0 and 300 mM Na⁺, recorded from excised inside-out patches from cells expressing wild-type (D) or M333A (E) Slo2.2. Pipette solution contained 150 mM KCl, 5 mM EDTA and 10 mM Hepes (pH 7.4) and bath solution contained 150 mM KCl, 10 mM Hepes (pH 7.4), and either 0 mM (top row) or 300 mM (bottom row) NaCl. The membrane voltage was held at 0 mV and stepped to voltages from −80 mV to 0 mV in 10 mV increments.

Figure 4. S1–S4 domain structure

(A) Stereo view of superposed open (blue) and closed (red) S1–S4 domains. Main-chain R.M.S.D is 0.83 Å. (B) Stereo view of open Slo2.2 S1–S4 domain shown in ribbon. Residues in S1–S4 that form inter-helix hydrogen bonds are shown with sticks and colored by atom.

Figure 5. Image analysis workflow

Representative images of Slo2.2 channels recorded in the presence of 20 mM, 40 mM, 80 mM, 160 mM and 300 mM Na⁺. Slo2.2 particles were automatically selected from the images (green circles). The extracted particles from the 20 mM, 40 mM, 80 mM and 160 mM Na⁺ images were combined into a single titration data set for 3D refinement, while the 300 mM Na⁺ data set was refined independently. Using the angles and orientations obtained from the 3D refinement, the particles from the titration data set and the 300 mM Na⁺ data set were classified into 10 classes. The closed classes are colored red and the open classes are colored blue.

Figure 6. Reproducibility of 3D classification

(A, C, E) Cryo-EM density maps of open Slo2.2 (A) and the two closed Slo2.2 classes with the most extreme rotations (C - closed class 1, E - closed class 9). (B, D, F) Fraction of particles in the open class (B) and the two closed Slo2.2 classes with the most extreme rotations (D - closed class 1 and F - closed class 9) from the titration data set is plotted as a function of Na⁺ concentration for 5 independent 3D refinement and classification runs. See also Figures S4 and S5.

Figure 7. Na⁺ titration of Slo2.2

(A–E) Representative cryo-EM images of Slo2.2 vitrified in the presence of 20 mM (A), 40 mM (B), 80 mM (C), 160 mM (D) and 300 mM Na⁺ (E). Particles marked with a red circle were classified as closed and those with a blue circle were classified as open. (F) Representative macroscopic current traces of intracellular Na⁺ activation. Currents were recorded using using the inside-out patch-recording configuration; pipette solution contained 150 mM KCl, 5 mM EDTA, and 10 mM Hepes (pH 7.4). Bath solution contained 150 mM KCl, 10 mM Hepes (pH 7.4), and 0 mM to 600 mM NaCl controlled by bath perfusion. Membrane voltage was held at 0 mV and stepped to −60 mV. (G) Variance (σ²)-mean current (<I>) relationship for traces shown in (F) and fit to a parabola (equation 1, methods). A fit to this equation yielded estimates of unitary conductance (i) and channel number (N) through application of equations 1 and 2 (methods). (H) Open probability deduced from data as in (F) is graphed as a function of intracellular Na⁺ (black, error bars represent SEM for 5 experiments). The structural open probability based on cryo-EM is plotted on the same graph (blue, error bars represent standard deviation for 5 independent classifications). A Hill function fit to the electrophysiology data yields a binding constant for Na⁺ of 0.20 ± 0.02 M, a Hill coefficient of 3.0 ± 0.5 and maximum value 0.68 ± 0.03. A Hill function fit to the structural data with a fixed maximum of 1.0 yields a binding constant of 0.24 ± 0.04 M and a Hill coefficient of 3.5 ± 1.3. See also Figure S6.