The Lipid Dependence of Purified and Reconstituted Kir2.1 and Kir2.2

Abstract

Ion channels are embedded in the membrane bilayer and are known to be regulated by their lipid environment. Insights on the structural basis of channel-lipid interactions have been gained by recent potassium channel crystal structures that reveal bound lipid or detergent molecules. However, efforts to define the lipid dependence of channel activity have been limited to cellular expression systems, in which the membrane composition cannot be fully controlled. We have expressed and purified functional human Kir2.1 and Kir2.2 from S. cerevisiae, and characterized the phospholipid dependence of channel activity in a liposomal 86Rb+ flux assay. Reconstituted Kir2.1 and Kir2.2 require incorporated PIP2 for activity and are maximally active in 0.1-1% PIP2 on a background of 3:1 POPE:POPG. This provides definitive evidence that eukaryotic Kir channels are directly activated by PIP2 without any intermediary components. Interestingly, Kir2.1 and Kir2.2 are minimally active in ∼1% PIP2 on a POPE (neutral) background, and are activated by increasing amounts of POPG (1 negative charge) or other anionic phospholipids. By contrast, the prokaryotic inward rectifier, KirBac1.1, shows no phospholipid dependence of activity, except potent inhibition by PIP2 (1), DGS-NTA, cardiolipin and oleoyl CoA. Our data suggest that the site of action for this secondary regulation by anionic phospholipids in Kir2.1 and Kir2.2 is distinct from the cytoplasmic PIP2 binding site. This study represents the first description of the lipid dependence of activity for recombinantly-expressed, purified eukaryotic ion channels in liposomes, and demonstrates that Kir2.1 and Kir2.2 have two lipid requirements for activity: a high affinity requirement that is specific for PIP2, and a low affinity requirement that is relatively non-specific for anionic phospholipids.1. D. Enkvetchakul, I. Jeliazkova, C. G. Nichols, J.Biol.Chem. 280, 35785 (2005).