Phospholipid Regulation of Purified and Reconstituted Human Inward Rectifier (Kir) Channels

Abstract

It is becoming increasingly clear that membrane lipids are critical determinants of ion channel function, but previous studies have provided limited and largely qualitative information on how lipids influence Kir channel function because they typically utilize cell-based systems where membrane composition is unknown and cannot be precisely controlled. Recent breakthroughs have enabled the purification of human Kir2.1 channels (D’Avanzo et al., Protein Expr Purif. 2010 May;71(1):115-21) that can now be reconstituted into liposomes of defined composition to determine quantitative and mechanistic regulation of channel activity by membrane lipids. With the use of 86Rb+ flux assays and patch clamp on giant proteoliposomes we now definitively show that these channels are directly activated by PI(4,5)P2 and that PI(4,5)P2 is absolutely required in the membrane for channel activity, with half maximal activity at ∼0.1% PI(4,5)P2. Activation of Kir2.1 by PIPs is also highly selective for PI(4,5)P2; PI(3,5)P2 does not activate channels and PI(3,4,5)P3 activates the channels minimally. We further demonstrate that Kir2.1 has a previously unresolved secondary non-specific requirement for anionic phospholipids. Kir2.1 channels are activated by PG, PS, PA, PI, or Cardiolipin with half-maximal activation at ∼5%, in the presence of 1% PI(4,5)P2, but not by PC, or EPC. No activation was observed by anionic phospholipids in the absence of PI(4,5)P2. Patch clamp analysis reveals that both unitary conductance and open probability are elevated by increasing membrane POPG concentration--unlike PI(4,5)P2 which only increases open probability. Collectively, these data demonstrate quantitative dependence of human Kir channel activity on PI(4,5)P2 in membranes of defined composition, and reveal a previously unrecognized secondary requirement for anionic lipids.