Dual-Mode Phospholipid Regulation of Human Inward Rectifying Potassium Channels

Abstract

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using (86)Rb(+) flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP(2) increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP(2), and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP(2) and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.

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

“…We also showed previously that Kir2.1 are very specifically activated by PI(4,5)P 2 , with only ϳ10% of maximal activity by PI(3,4,5)P 3 and little or no activation by the remaining PIPs (2). Furthermore, in the presence of high concentrations of anionic phospholipids such as phosphatidylglycerol, other PIPs can competitively inhibit PI(4,5)P 2 -dependent channel activity (5). This suggests that the various PIPs may bind to the same general location in Kir2.1 channels, but does not explain why they do not equivalently trigger channel activation.…”

“…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.…”

“…Giant liposomes were prepared using a dehydration-rehydration method in a manner similar to that described previously (2,5). For patch clamp, giant proteoliposomes were pipetted onto a glass coverslip in an oil-gate chamber (22) and allowed to settle for ϳ5 min before starting the solution exchange to wash away debris.…”

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.…”