Cooperative Gating of a K+ Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy.

Abstract

Lipids adhere to membrane proteins to stimulate or suppress molecular and ionic transport and signal transduction. Yet, the molecular details of lipid-protein interaction and their functional impact are poorly characterized. Here we combine NMR, coarse-grained molecular dynamics (CGMD), and functional assays to reveal classic cooperativity in the binding and subsequent activation of a bacterial inward rectifier potassium (Kir) channel by phosphatidylglycerol (PG), a common component of many membranes. Past studies of lipid activation of Kir channels focused primarily on phosphatidylinositol bisphosphate, a relatively rare signaling lipid that is tightly regulated in space and time. We use solid-state NMR to quantify the binding of unmodified 13C-PG to the K + channel KirBac1.1 in liposomes. This specific lipid-protein interaction has a dissociation constant ( K d) of ∼7 mol percentage PG (Χ PG) with positive cooperativity ( n = 3.8) and approaches saturation near 20% Χ PG. Liposomal flux assays show that K + flux also increases with PG in a cooperative manner with an EC 50 of ∼20% Χ PG, within the physiological range. Further quantitative fitting of these data reveals that PG acts as a partial (80%) agonist with fivefold K + flux amplification. Comparisons of NMR chemical shift perturbation and CGMD simulations at different Χ PG confirm the direct interaction of PG with key residues, several of which would not be accessible to lipid headgroups in the closed state of the channel. Allosteric regulation by a common lipid is directly relevant to the activation mechanisms of several human ion channels. This study highlights the role of concentration-dependent lipid-protein interactions and tightly controlled protein allostery in the activation and regulation of ion channels.