Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels

All members of the inward rectifiier K+ (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K-ATP channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K-ATP channel activity, respectively. GFP-tagged Kir6:2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic ml receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues similar to306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, 1309, W311 and F315, consistent with residues lying on one side of a a-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.