The conserved potassium channel filter can have distinct ion binding profiles: structural analysis of rubidium, cesium, and barium binding in NaK2K

Abstract

Potassium channels are highly selective for K(+) over the smaller Na(+). Intriguingly, they are permeable to larger monovalent cations such as Rb(+) and Cs(+) but are specifically blocked by the similarly sized Ba(2+). In this study, we used structural analysis to determine the binding profiles for these permeant and blocking ions in the selectivity filter of the potassium-selective NaK channel mutant NaK2K and also performed permeation experiments using single-channel recordings. Our data revealed that some ion binding properties of NaK2K are distinct from those of the canonical K(+) channels KcsA and MthK. Rb(+) bound at sites 1, 3, and 4 in NaK2K, as it does in KcsA. Cs(+), however, bound predominantly at sites 1 and 3 in NaK2K, whereas it binds at sites 1, 3, and 4 in KcsA. Moreover, Ba(2+) binding in NaK2K was distinct from that which has been observed in KcsA and MthK, even though all of these channels show similar Ba(2+) block. In the presence of K(+), Ba(2+) bound to the NaK2K channel at site 3 in conjunction with a K(+) at site 1; this led to a prolonged block of the channel (the external K(+)-dependent Ba(2+) lock-in state). In the absence of K(+), however, Ba(2+) acts as a permeating blocker. We found that, under these conditions, Ba(2+) bound at sites 1 or 0 as well as site 3, allowing it to enter the filter from the intracellular side and exit from the extracellular side. The difference in the Ba(2+) binding profile in the presence and absence of K(+) thus provides a structural explanation for the short and prolonged Ba(2+) block observed in NaK2K.

Figure 1.

Overall structure of the NaK2K–K⁺ complex in open conformation with the front and back subunits removed for clarity. A magnified view of its selectivity filter (boxed) is shown. Electron density (blue mesh) from the F_o–F_c ion omit map (4σ) shows K⁺ ions (modeled as green spheres) in the filter and water molecules (modeled as red spheres) at site 0. The four ion-binding sites within the filter are labeled 1–4 from top to bottom.

Figure 2.

Rb⁺ and Cs⁺ binding in NaK2K. (A) F_o–F_c ion omit map (6σ, blue mesh) of the NaK2K–Rb⁺ complex and the one-dimensional electron density profile (from a 1.85-Å ion omit map) along the central axis of the filter. The electron density distribution of K⁺ ions in the NaK2K–K⁺ complex is also plotted (in gray) for comparison (and for all other one-dimensional profiles). The Rb⁺ ions are modeled as purple spheres. (B) Anomalous difference Fourier map (7σ, red mesh) of the NaK2K–Rb⁺ complex and the one-dimensional anomalous scattering profile at the selectivity filter region. (C) F_o–F_c ion omit map (6σ) of the NaK2K–Cs⁺ complex and the one-dimensional electron density profile along the filter. The Cs⁺ ions are modeled as cyan spheres. The density at site 4 is modeled as a Na⁺ ion (orange sphere). (D) Anomalous difference Fourier map (7σ) of the NaK2K–Cs⁺ complex and the one-dimensional anomalous scattering profile at the selectivity filter region.

Figure 3.

Rb⁺ and Cs⁺ permeation in NaK2K. (A) Single-channel traces of NaK2K at ±80 mV with a symmetrical 150 mM RbCl and its I-V curve (n = 5 measurements). Currents were recorded using giant liposome patch clamping. The same method was used in all other recordings. The arrows indicate the baseline. (B) Single-channel traces of NaK2K with 150 mM of a mixture of Rb⁺ and K⁺ and its I-V curve (n = 5 measurements). The channel is slightly selective for K⁺ with a reversal potential of ∼10 mV. (C) The I-V curves of NaK2K in the presence and absence of Cs⁺. The pipette solution contains 150 mM KCl. The bath solution contains 150 mM KCl (black trace), 135 mM KCl and 15 mM CsCl (blue trace), or 150 mM CsCl (red trace). For a better assessment of reversal potential, each I-V curve was obtained from the mean of 20 current traces recorded using a patch containing multiple channels with voltage ramps from −100 to 100 mV over a 400-ms duration. Error bars represent SEM.

Figure 4.

Ba²⁺ block in NaK2K. (A) Internal Ba²⁺ block in the presence and absence of external K⁺. The nonconducting dwell time histograms with exponential fits for each recording are shown beside each trace. Only the patch that contains a channel with its intracellular side facing the bath solution was used in the recording. (B) External Ba²⁺ block in the presence of 5 mM external K⁺. Only the patch that contains a channel with its extracellular side facing the bath solution was used in the recording.

Figure 5.

Ba²⁺ binding in the NaK2K filter in the presence of K⁺. (A) F_o–F_c ion omit map (6σ) of the NaK2K–Ba²⁺ complex and the one-dimensional electron density profile along the filter. The Ba²⁺ ions are modeled as yellow spheres. The density at sites 1, 2, and 4 is modeled as K⁺ ions (green spheres) for simplicity. The inset (boxed) is a magnified view of the external Ba²⁺ with the F_o–F_c ion omit map contoured at 3σ to show the density of water molecules. (B) Anomalous difference Fourier map (7σ) of the NaK2K–Ba²⁺ complex and the one-dimensional anomalous scattering profile at the selectivity filter region.

Figure 6.

Ba²⁺ binding in the NaK2K filter in the absence of K⁺. (A) F_o–F_c ion omit map (6σ) of the NaK2K–Ba²⁺ complex in NaCl and the one-dimensional electron density profile along the filter. The Ba²⁺ ions are modeled as yellow spheres. Site 2 density is modeled as water (red spheres), and site 4 density is modeled as a Na⁺ ion (orange spheres). The inset (boxed) is a magnified view of site 4 Na⁺ with the F_o–F_c ion omit map contoured at 3σ to show the density of the cavity water molecule. (B) Anomalous difference Fourier map (7σ) of the NaK2K–Ba²⁺ complex in NaCl and the one-dimensional anomalous scattering profile at the selectivity filter region.

Figure 7.

Cartoon representation of the fast (no external K⁺) and slow (with external K⁺) Ba²⁺ blocks from the intracellular side with an outward electrochemical driving force.

Figure 8.

Comparison of the structural snapshot of the Ba²⁺/K⁺ binding profile in NaK2K filter with the multi-ion binding sites deduced from the Ba²⁺ block study in BK channels. The sizes of the green spheres are scaled to represent K⁺ affinity (occupancy). The site 4 ion is represented by a gray sphere to indicate the nonselective nature of the site.