Direct Modulation of Kir Channel Gating by Membrane Phosphatidylinositol 4,5-Bisphosphate

Abstract

Multiple ion channels have now been shown to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) at the cytoplasmic face of the membrane. However, direct evidence for a specific interaction between phosphoinositides and ion channels is critically lacking. We reconstituted pure KirBac1.1 and KcsA protein into liposomes of defined composition (3:1 phosphatidylethanolamine:phosphatidylglycerol) and examined channel activity using a 86 Rb ؉ uptake assay. We demonstrate direct modulation by PIP 2 of KirBac1.1 but not KcsA activity. In marked contrast to activation of eukaryotic Kir channels by PIP 2 , KirBac1.1 is inhibited by PIP 2 incorporated in the membrane (K1 ⁄ 2 ‫؍‬ 0.3 mol %). The dependence of inhibition on the number of phosphate groups and requirement for a lipid tail matches that for activation of eukaryotic Kir channels, suggesting a fundamentally similar interaction mechanism. The data exclude the possibility of indirect modulation via cytoskeletal or other intermediary elements and establish a direct interaction of the channel with PIP 2 in the membrane.Phosphoinosotides constitute a major group of signaling molecules in eukaryotic membranes (1, 2) and modulate an ever growing list of ion channels, whether by application of exogenous phospholipids to the cytoplasmic membrane surface or by manipulation of endogenous phospholipids (3-12). However, the nature of the phosphoinositide-channel interaction remains elusive. For one extensively studied group, the inward rectifier K (Kir) channels, there is an emerging consensus that a direct interaction occurs between cytoplasmic domains of the channel and inositol headgroups, based on electrophysiological analysis (5, 13-16) and biochemical analysis of isolated channel domains (5,17,18). Direct interaction of functional channels with phospholipids in the membrane has been difficult to demonstrate unequivocally, and without this, quantification of the dose-response relationships for channel modulation by phospholipids is obviated, and further mechanistic understanding is limited (19, 20).The recent cloning and crystallization (21), as well as functional analysis of KirBac1.1 channels reconstituted in lipid membranes (22), provides the opportunity to examine channel activity using a highly purified protein preparation in membranes of defined composition and permits direct test of the nature of the channel-phosphoinositide interaction. EXPERIMENTAL PROCEDURESMethods are essentially as described previously (22). KcsA and KirBac1.1 in pQE60 vector were expressed in BL21* (DE3) cells after induction with isopropyl ␤-D-thiogalactopyranoside. Bacteria were lysed by sonication, incubated 2-4 h with 30 mM decylmaltoside (Anatrace), then centrifuged at 30,000 ϫ g for 30 min, and the supernatant was applied to a cobalt affinity column. The column was washed with 20 -30 volumes of wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM KCl, 10 mM imidazole, and 5 mM decylmaltoside) and eluted with 1-2 ml of wash buffer containing 500 mM imidazole. Protein was concentrated u...

Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides

“…Disruption of this binding site counters PI3-K actions on ENaC activity, as well as disrupts increases in ENaC open probability in response to addition of exogenous PIP 3 . These findings thus demonstrate that similar to its actions on Kir channels (21,45,61,62), PIP 3 physically associates with ENaC to affect open probability.…”

Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels

“…Prokaryotic Kir channels are inhibited by PIP 2 (11,12), whereas Kir channels from all eukaryotic cells studied to date are activated by PIP 2 (2,6,10,13). This is an interesting difference, but PIP 2 is not present in the prokaryotic cell membrane (2,6), and thus its inhibitory effect on bacterial Kir channels lacks physiological relevance.…”

Energetics and Location of Phosphoinositide Binding in Human Kir2.1 Channels

“…Members of this family are directly regulated by phosphoinositides (PIPs) even in the absence of other proteins or downstream signaling pathways (1)(2)(3)(4). Some members show variable specificity for the activating PIP, but all eukaryotic Kir channels are activated by PI(4,5)P 2 (2), and members of the Kir2 subfamily, including human Kir2.1 channels, are quite selectively activated by this ligand (2,5).…”