Structural and Functional Determinants of Conserved Lipid Interaction Domains of Inward Rectifying Kir6.2 Channels

Abstract

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 δ1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor–mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple β-strands and a conserved α-helix (residues ∼306–311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a α-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.