Direct and Specific Activation of Human Inward Rectifier K+ Channels by Membrane Phosphatidylinositol 4,5-Bisphosphate

Abstract

Many ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ), but studies examining the PIP 2 dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP 2 dependence of purified human Kir2.1 and Kir2.2 activity using 86 Rb ؉ flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP 2 and that PIP 2 is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP 2 levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP 2 , and open probability increases to ϳ0.4 at 1% PIP 2 . Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP 2 ; PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P 3 causes minimal activity. The PIP 2 dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP 2 . This raises the interesting hypothesis that PIP 2 activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP 2 in the eukaryotic cell membrane.

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.…”

Focus on Kir7.1: physiology and channelopathy

“…55 One of the best characterized lipid modulators of K ir channel activity is membrane PIP2 (phosphatidylinositol 4, 5-bisphosphate). [56][57][58][59][60] PIP2 is found principally in the cytoplasmic leaflet of the plasma membrane that comprises »1 % of plasma membrane phospholipids. 57 Membrane PIP2 abundance is a dynamic entity and is precisely controlled by lipid kinases, phospholipases, and phosphatases.…”

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). To understand why Kir2.1 channels are selectively activated by PI(4,5)P 2 over other PIPs, it is necessary to identify the location and structure of the PIP binding site(s).…”

“…WT and mutant Kir2.1-FLAG-His 8 fusion proteins were expressed in and purified from the FGY217 strain of Saccharomyces cerevisiae as described previously (2,5,20,45). Mutagenesis was performed using QuikChange II site-directed mutagenesis kits (Stratagene) and verified by sequencing.…”

“…Channel activity and lipid dependence were assessed by measuring 86 Rb ϩ uptake into proteoliposomes containing reconstituted Kir2.1 protein as described previously (2,20,45). Valinomycin was used to measure maximal 86 Rb ϩ uptake.…”