High-resolution structure of the open NaK channel

Abstract

We report the crystal structure of the nonselective cation channel NaK from Bacillus cereus at a resolution of 1.6 A. The structure reveals the intracellular gate in an open state, as opposed to the closed form reported previously, making NaK the only channel for which the three-dimensional structures of both conformations are known. Channel opening follows a conserved mechanism of inner helix bending using a flexible glycine residue, the gating hinge, seen in MthK and most other tetrameric cation channels. Additionally, distinct inter and intrasubunit rearrangements involved in channel gating are seen and characterized for the first time along with inner helix twisting motions. Furthermore, we identify a residue deeper within the cavity of the channel pore, Phe92, which is likely to form a constriction point within the open pore, restricting ion flux through the channel. Mutating this residue to alanine causes a subsequent increase in ion-conduction rates as measured by (86)Rb flux assays. The structures of both the open and closed conformations of the NaK channel correlate well with those of equivalent K(+) channel conformations, namely MthK and KcsA, respectively.

Figure 1

Structure of NaKNΔ19 in an open conformation. (a) Overall structure of NaKNΔ19 with the front subunit removed for clarity. Selectivity filter residues, Gly87 and Phe92 are colored yellow, red and green, respectively. M1=outer helix, M2=inner helix, and P=pore helix b) Superimposition of the structures of NaKNΔ19 (blue) and NaK (red, PDB code 2AHY) with its M0 helix removed, viewed from the plane of the membrane with proximal and distal subunits removed and c) from the intracellular side. d) Conformational change of NaK inner helices from the closed (red cylinder) to open state (blue cylinder) involves a 34° bending and a 45° twisting around the helical axis. e) Comparison of NaK with KcsA (left) and MthK (right). Superimposition of the closed NaK structure (red, PDB code 2AHY) with KcsA (yellow, PDB code 1K4C) viewed from the intracellular side along 4-fold axis and the equivalent superimposition of the open NaKNΔ19 structure (blue) with the MthK pore (orange, PDB code1LNQ) are shown.

Figure 2

Analysis of inter and intra-subunit interactions in the closed (a) and open (b) structures of the NaK channel. The numbers in the boxed regions in both structures correspond to the zoom-in panels 1-3 for the closed structure and 1-2 for the open NaK structure. Zoom 1 in both cases highlights intra-subunit interactions between outer (M1) and inner (M2) helices. Zoom 2 highlights inter-subunit interactions between inner helices of two neighboring subunits. Zoom 3 shows an intracellular view of the interactions involved in the bundle crossing formation in the closed structure.

Figure 3

Effect of the size of the ion conduction pathway size on ion conduction in NaK. a) Surface representation of the ion conduction pathway of the NaKNΔ19 open pore and b) its Phe92Ala mutant. c) ⁸⁶Rb flux assay showing time dependent Rb⁺ influx in liposomes reconstituted with NaKNΔ19 (Red trace) and the Phe92Ala mutant (black trace). Both proteins were reconstituted in liposomes loaded with KCl at the same concentration. Liposomes with no reconstituted protein were used as a control (blue trace).