Structural analysis of ion selectivity in the NaK channel

Abstract

Here we present a detailed characterization of ion binding in the NaK pore using the high-resolution structures of NaK in complex with various cations. These structures reveal four ion binding sites with similar chemical environments but vastly different ion preference. The most nonselective of all is site 3, which is formed exclusively by backbone carbonyl oxygen atoms and resides deep within the selectivity filter. Additionally, four water molecules in combination with four backbone carbonyl oxygen atoms are seen to participate in K(+) and Rb(+) ion chelation, at both the external entrance and the vestibule of the NaK filter, confirming the channel's preference for an octahedral ligand configuration for K(+) and Rb(+) binding. In contrast, Na(+) binding in the NaK filter, particularly at site 4, utilizes a pyramidal ligand configuration that requires the participation of a water molecule in the cavity. Therefore, the ability of the NaK filter to bind both Na(+) and K(+) ions seemingly arises from the ions' ability to use the existing environment in unique ways, rather than from any structural rearrangements of the filter itself.

1

Na⁺ binding in the NaK filter (a) 2F_o-F_c ion omit map contoured at 1.5σ showing density (blue mesh) for Na⁺ ions, water molecules in the vestibule and central cavity, and the contaminating ion at site 3 (green, red, and orange spheres, respectively). The front and back subunits of the NaK model have been removed for clarity in most figures. (b) Comparison of Na⁺ binding in NaK at site 4 (upper) and the 3′ side of the guanine tetraplex (lower) showing Na⁺ binding in plane with 4 of its ligand, with a water molecule sitting axial to it. (c) 2F_o-F_c ion omit map contoured at 2.5σ (blue mesh, upper) showing football shape of electron density at site 3 of NaKNΔ19 in high Na⁺ (500mM); and the F_o-F_c map with Cs⁺ at site 3 contoured at 4 σ (red mesh, lower) showing density that likely arises from a contribution of Na⁺ present on both the upper and lower edges of site 3. (d) Suggested mode of Na⁺ binding at the upper and lower edges of site 3.

2

K⁺ and Rb⁺ binding in the NaK selectivity filter. The 2F_o-F_c ion omit maps (1.5σ) show electron density of ion binding in the K⁺ complex (a) and Rb⁺ complex (b) of NaKNΔ19. K⁺ and Rb⁺ ions are colored green with water molecules represented as red spheres. c) The maintenance of an octahedral ligand arrangement in the K⁺ complex, which also holds true for the Rb⁺ complex. Oxygen atoms from the front and back subunits chelating the ions are shown as red spheres. d) F_{Cs soak} − F_K difference map contoured at 10 σ showing Cs⁺ binding at site 3.

3

Ca²⁺ binding in the NaK selectivity filter. (a) 2F_o-F_c ion omit map (1.5σ) of the NaKNΔ19-Na⁺ complex co-crystallized with 10mM CaCl₂, showing electron density for a Ca²⁺ ion (purple sphere) at the external entrance. Na⁺ and the contaminant at site 3 are modeled as green and orange spheres, respectively. Electron density for the protein backbone is also shown as a blue mesh. (b) F_{0.5mM Ca} − F_{0mM Ca} (upper) and F_{5mM Ca²⁺} − F_{0mM Ca²⁺} (lower) difference map contoured at 10 σ showing increasing intensity of Ca²⁺ binding upon increasing Ca²⁺ concentration. Dotted lines indicate the position of central axis along which the 1-D electron density was calculated. (c) 1-D electron density profile of Ca²⁺ binding at the external entrance showing increased Ca²⁺ binding at increasing concentrations. The control is from a difference map between two native crystals grown in the absence of Ca²⁺. (d) Occupancy of Ca²⁺ binding at the external site was calculated by integration of the peaks in 1-D electron density profile and normalizing them against the 10 mM Ca²⁺ peak. The plot of occupancy against Ca²⁺ concentration give rise to a K_d value of about 1.8 mM upon fitting the curve to a single component Langmuir equation.

4

Ba²⁺ binding in NaK filter. a) F_{Ba soak} − F_K difference map (blue mesh) contoured at 10 σ showing the two Ba²⁺ binding sites. (b) 2F_o-F_c ion omit maps of Ba²⁺-soaked crystal contoured at 1.5 σ. The electron density at external entrance and site 3 was modeled as Ba²⁺ (orange spheres) and water molecules are modeled as red spheres. (c) Ba²⁺ (left) and Ca²⁺ (right) blocking of ⁸⁶Rb influx in liposomes loaded with NaCl.

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Comparison of the selectivity filter structures of NaK and KcsA and between various ion complexes of NaK. a) Superimposition of the NaK selectivity filter (yellow) in complex with K⁺ with that of KcsA (green, PDB code 1K4C). b) Superimposition of the selectivity filters of the NaKNΔ19 channel in complex with Na⁺ (yellow), K⁺ (green), and Rb⁺ (orange).