Probing the Interaction of Charged Lipids with the Potassium Channel KcsA

Abstract

The activity of integral membrane proteins has long been known to be tightly coupled to the lipid composition of the surrounding lipid bilayer. More recently though the presence of non-annular lipid binding sites have been shown to play a key role in the regulation of membrane channels. In particular recent fluorescence studies have revealed that gating of the potassium channel KcsA is highly dependent on the binding of anionic lipids to three or more non-annular lipid binding sites at the lipid protein interface1. Here we present solid-state NMR studies on KcsA reconstituted into charged lipid bilayers composed of POPC/POPG. These studies are allowing us to investigate the nature of the interaction between the surrounding lipid and these binding sites.Employing 1H-31P saturation transfer MAS NMR2 we have been able to probe the proximity and rate of exchange of lipid in close proximity to the KcsA. A significant attenuation of the POPC resonance was observed upon the saturation of amide protons suggesting that POPC populates the annular sites of KcsA and is in relatively fast exchange with the bulk lipid. In contrast no such attenuation was observed for the POPG, which in light of earlier fluorescence studies suggests that the POPG remains resident at the lipid protein interface and does not readily exchange with the bulk lipid. Preliminary heteronuclear correlation spectra in conjunction with T2 filtering are beginning to provide us with insights into the types of residues involved in this interaction.1) P. Marius et al., Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening. Biophysical Journal 2008 (94)1689-98.2) O. Soubias et al., Evidence for lipid specificity in lipid-rhodopsin interactions Journal of Biological Chemistry 2006 (281)33233-41.