Plants acquire potassium (K⁺) ions for cell growth and movement via regulated diffusion through K⁺ channels. Here, we present crystallographic and functional data showing that the K⁺ inward rectifier KAT1 (K⁺Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H⁺-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K⁺ uptake in plant cells.

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K⁺ channels, Kcv and KcsA, as well as a recently discovered dual-topology F⁻ channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels.

Many potassium channels show voltage-dependent gating without a dedicated voltage sensor domain. This is not fully understood yet, but often explained by voltage-induced changes of ion occupation in the five distinct K⁺ binding sites in the selectivity filter. To better understand this mechanism of filter gating we measured the single-channel current and the rate constant of sub-millisecond channel closure of the viral K⁺ channel Kcv_NTS for a wide range of voltages and symmetric and asymmetric K⁺ concentrations in planar lipid membranes. A model-based analysis employed a global fit of all experimental data, i.e., using a common set of parameters for current and channel closure under all conditions. Three different established models of ion permeation and various relationships between ion occupation and gating were tested. Only one of the models described the data adequately. It revealed that the most extracellular binding site (S0) in the selectivity filter functions as the voltage sensor for the rate constant of channel closure. The ion occupation outside of S0 modulates its dependence on K⁺ concentration. The analysis uncovers an important role of changes in protein flexibility in mediating the effect from the sensor to the gate.

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K⁺ channels, Kcv_S and Kcv_NTS. These small viral encoded K⁺ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K⁺ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in Kcv_NTS and 40% in Kcv_S. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in Kcv_S. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.