The Molecular Mechanisms of Cholesterol Regulation of Kir Channels Revealed by Direct and Quantitative Approaches

Abstract

Inwardly rectifying potassium (Kir) channels regulate cell excitability in multiple tissues and are key modulators of cardiac and vascular electrical activity and contractility. Channel activity is regulated by various ligands including membrane lipids. Phosphoinositol-4,5-bisphosphate (PIP2) is the absolutely required agonist for all Kir channel subfamily members while cholesterol exhibits mixed effects depending on the subfamily; it inhibits Kir2 but increases the open probability of Kir3 channels. In order to unambiguously determine cholesterol binding sites as well as regulatory mechanisms, quantitative and more direct approaches have been developed. First, an in vitro functional assay system has been established to measure Kir channel activity in the presence of cholesterol as high as 40 mol%. Second, photocrosslinkable cholesterol mimetics (LKM38) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches have been developed to directly detect channel residues in close proximity to cholesterol within membranes. With these tools, we demonstrate both concentration-dependent and non-competitive Kir2 channel inhibition by cholesterol, suggesting that cholesterol has binding site(s) not affecting PIP2 binding to the channel. Further, we detect two cholesterol-interacting sites in MS experiments, one previously identified[1], and a novel site that has not been suggested. The same approaches will be repeated for Kir3 channels and subsequent docking and molecular dynamics simulations may enlighten the molecular mechanisms of the differential cholesterol regulations of Kir channels. 1. Rosenhouse-Dantsker, A., et al., Identification of novel cholesterol-binding regions in Kir2 channels. J Biol Chem, 2013. 288(43): p. 31154-64.