Inward-rectifier K+ (Kir) channels are tetrameric ligand-gated ion channels. These channels have high affinity for anionic lipids within their natural bilayers, which are often their native activating ligands. These “classical” Kir channels conduct inward K+ current in response to anionic lipid association, regulating the resting membrane potential in excitable cells. Noted channelopathies include forms of heart disease, psychiatric disorders, and alcoholism. The prokaryotic channel KirBac1.1 shares its fold and regulatory mechanisms with homologous human channels. Using solid-state NMR (SSNMR), SSNMR-driven simulated annealing structure calculations, and coarse-grain molecular dynamics (CG-MD) we resolved an intricate allosteric network between the inner gate, outer gate, and intracellular Kir domain. This network is governed by positive anionic lipid-loading allostery. We present a SSNMR titration of 13C-phosphadityl glycerol (PG) in the presence of KirBac1.1 which reveals sigmoidal lipid binding with a Kd of 7% bilayer PG content and a Hill Coefficient of 4 at physiological temperature. This finding indicates that channel activity can be regulated by the cell through control of bilayer composition. To borrow concepts from enzymology, this implies that the closed/inactivated state of the channel corresponds to a tense (T) state, and the open/activated state of the channel is the relaxed (R) state. Next, we identify the locus of PG binding and the activated channel structure. Solvent accessibility measurements and three-dimensional 13C-13C-13C dipole-assisted rotational resonance (DARR) experiments were used to solve the structure of KirBac1.1 in both the R, activated and T, inactivated states. This effort established a new set of water-edited restraints for SSNMR-driven simulated annealing. We then leveraged these techniques to solve the structure of a cholesterol-inactivated T state of the channel, found in samples containing 2-13C-acetate cholesterol and either 1,3-13C-glycerol or 2-13C-glycerol KirBac1.1. This effort uncovered a conserved Kir channel cholesterol recognition motif occupied by a persistent cholesterol dimer.

Inward-rectifier K+ channel transmembrane and positive lipid-loading allostery observed by solid-state NMR

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