Inwardly rectifying potassium (Kir) channels regulate multiple tissues. All Kir channels require interaction of phosphatidyl-4,5-bisphosphate (PIP2) at a crystallographically identified binding site, but an additional nonspecific secondary anionic phospholipid (PL(−)) is required to generate high PIP2 sensitivity of Kir2 channel gating. The PL(−)-binding site and mechanism are yet to be elucidated. Here we report docking simulations that identify a putative PL(−)-binding site, adjacent to the PIP2-binding site, generated by two lysine residues from neighbouring subunits. When either lysine is mutated to cysteine (K64C and K219C), channel activity is significantly decreased in cells and in reconstituted liposomes. Directly tethering K64C to the membrane by modification with decyl-MTS generates high PIP2 sensitivity in liposomes, even in the complete absence of PL(−)s. The results provide a coherent molecular mechanism whereby PL(−) interaction with a discrete binding site results in a conformational change that stabilizes the high-affinity PIP2 activatory site.

Crystallography has provided invaluable insights to ion channel selectivity and gating, but to advance understanding to a new level, dynamic views of channel structures within membranes are essential. We labeled tetrameric KirBac1.1 potassium channels with single donor and acceptor fluorophores at different sites, and examined structural dynamics within lipid membranes by single molecule FRET. We found that the extracellular region is structurally rigid in both closed and open states, whereas the N-terminal slide helix undergoes marked conformational fluctuations. The cytoplasmic C-terminal domain fluctuates between two major structural states both of which become less dynamic and move away from the pore axis and away from the membrane in closed channels. Our results reveal mobile and rigid conformations of functionally relevant KirBac1.1 channel motifs, implying similar dynamics for similar motifs in eukaryotic Kir channels and for cation channels in general.

Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+, but not Na+, ions are perfectly coordinated. Using single molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentrations. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels non-selective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild type KirBac1.1 channels are K+-selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, while K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

Inward rectifier potassium (Kir) channels are physiologically regulated by a wide range of ligands that all act on a common gate, although structural details of gating are unclear. Here we show, using small molecule fluorescent probes attached to introduced cysteines, the molecular motions associated with gating of KirBac1.1 channels. The accessibility of the probes indicates a major barrier to fluorophore entry to the inner cavity. Changes in FRET between fluorophores attached to KirBac1.1 tetramers show that PIP2-induced closure involves tilting and rotational motions of secondary structural elements of the cytoplasmic domain that couple ligand binding to a narrowing of the cytoplasmic vestibule. The observed ligand-dependent conformational changes in KirBac1.1 provide a general model for ligand-induced Kir channel gating at the molecular level.