Symmetry Reduction in a Hyperpolarization-Activated Homotetrameric Ion Channel

Abstract

Plants obtain nutrients from the soil via transmembrane transporters and channels in their root hairs, from which ions radially transport in toward the xylem for distribution across the plant body. We determined structures of the hyperpolarization-activated channel AKT1 from Arabidopsis thaliana, which mediates K⁺ uptake from the soil into plant roots. These structures of AtAKT1 embedded in lipid nanodiscs show that the channel undergoes a reduction of C4 to C2 symmetry, possibly to regulate its electrical activation.

Figure 1.

CryoEM structure and functional state of AtAKT1 in a lipid bilayer. (A) Molecular model of the AtAKT1 homotetramer with a single subunit colored by domain. (B) Sharpened electrostatic potential map, colored by domain as in (A) with low-pass filtered envelope shown to depict the nanodisc and poorly resolved ankyrin repeats. (C) The deactive voltage sensing domain with charge transfer center residues shown. (D) Pore profile of the closed channel with pore-lining residues on the P-loop and S6 shown including the selectivity filter near the extracellular side, and activation gate at the intracellular side. The pore radius is shown plotted with respect to the distance along the channel axis, calculated using MOLE. (E) Atomic model of the selectivity filter and K⁺ ion superpositions, with sharpened electrostatic potential map shown zoned 2.5 Å around the atoms of interest. (F) Residues that form the selectivity filter from AtAKT1 and other K⁺ channels, highlighting the signature GYG motif.

Figure 2.

Symmetry reduction in AtAKT1 and its implications for voltage activation. (A) The C-linker adopts two different conformations in the C2-symmetric structure. In one conformation, the linker is kinked (left box), forming a nexus with S4 and S6. In the adjacent subunit, the C-linker is flattened by insertion of the pre-VSD N-terminal helix, breaking most atomic interactions in the nexus (right box). (B) Graphical model of how N-helix insertion flattens the C-linker (represented by solid arrows) and potentially interferes with the downward translocation of S4 during membrane hyperpolarization (represented by dashed arrows). (C) Orthogonal views of overlaid AKT1 kinked and flat subunits (left), and apo- and holo-CNGA1 (right) (PDBs: 7LFT and 7LFW, respectively) highlighting differences in C-linker and CNBD conformations. (D) Atomic interactions between the N-helix, VSD, C-linker, and pore domain. (E) Three conformations of the CNBHD domains are recovered from 3D classification (right), the C4 class at ∼2.6 Å displayed at top, mid-C2 class at ∼2.8 Å in the center (used for model building and atomic interpretation), and ultra-C2 class at ∼4.5 Å on the bottom, all viewed at low contour. The soluble region in the mid-C2 class was resolved to high resolution and was used to build an atomic model of AKT1, shown on the left. The degree of asymmetry is quantified by the distances between D337 on opposite C-linkers. (F) Orthogonal views of the mid-C2 map, displayed at low contour to show the ankyrin domains in two distinct configurations: one pair exhibits extensive interdomain interactions, while the neighboring pair does not touch.