Identification of the Regulatory Anionic Lipid Site in Kir Channels

Inwardly rectifying potassium channels (Kir) are regulated by multiple factors, including multiple lipids. In addition to a specific requirement for phosphatidyl-4,5-bisphosphate (PI(4,5)P2) for channel activity of Kir2.1, analysis of purified proteins reconstituted into liposomes has revealed a secondary requirement for non-specific anionic lipids, which increase PI(4,5)P2 sensitivity by ∼100 fold [Cheng et al. 2011, Biophys. J. 100, 620-628]. Recent crystal structures of eukaryotic Kir channels in complex with PI(4,5)P2 reveal a common PI(4,5)P2 binding site [Hansen et al, 2011, Nature. 477, 495-498; Whorton & MacKinnon, 2011, Cell. 147, 199-208], but they have not identified the synergistic anionic lipid site.We have performed extensive docking simulations to identify potential interaction sites of different phospholipids with Kir2.1 channels. These simulations indicate two distinct binding sites; a high affinity site that corresponds to the crystallographic PI(4,5)P2 binding pocket and a lower affinity site, involving two lysine residues further towards the periphery of the cytoplasmic domain, that may correspond to the secondary anionic lipid site. When the two lysine residues are mutated to cysteine, channel activity is essentially abolished, even in the presence of PIP2. Cysteine modification of these residues by decyl-MTS, which essentially provides a ‘lipid tether' to the residue, restores channel activity in the presence of low levels of PIP2. These results point strongly to the identified site as being the site for non-specific anionic lipid interaction, and support a model in which the anionic lipid interaction (or ‘lipid tethering') pulls the Kir domain towards the membrane, facilitating PI(4,5)P2-mediated channel opening.