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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Regulation of Kir channels in bovine retinal pigment epithelial cells by phosphatidylinositol 4,5-bisphosphate

Departments of ¹Ophthalmology and Visual Sciences and ²Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

Submitted 9 June 2009 ; accepted in final form 29 July 2009

The inwardly rectifying K⁺ (Kir) current in mammalian retinal pigment epithelial (RPE) cells, which is largely mediated by Kir7.1 channels, is stable in cells dialyzed with MgATP but runs down when intracellular ATP is depleted. A potential mechanism for this rundown is a decrease in phosphatidylinositol 4,5-bisphosphate (PIP₂) regeneration by ATP-dependent lipid kinases. Here, we used the whole cell voltage-clamp technique to investigate the membrane PIP₂ dependence of Kir channels in isolated bovine RPE cells. When RPE cells were dialyzed with ATP-free solution containing PIP₂ (25–50 µM), rundown persisted but was markedly reduced. Removal of Mg²⁺ from the pipette solution also slowed rundown, indicating that elevated intracellular Mg²⁺ concentration contributes to rundown. Cell dialysis with the PIP₂ scavenger neomycin in MgATP solution diminished Kir current in a voltage-dependent manner, suggesting that it acted at least in part by blocking the Kir channel. Kir current in MgATP-loaded cells was partially inhibited by bath application of quercetin (100 µM), phenylarsine oxide (100 µM), or wortmannin (50 µM), inhibitors of phosphatidylinositol (PI) kinases, and was completely inhibited by cell dialysis with 2 mM adenosine, a PI4 kinase inhibitor. Both LY-294002 (100 µM), an inhibitor of PI3 kinases, and its inactive analog LY-303511 (100 µM) rapidly and reversibly inhibited Kir current, suggesting that these compounds act as direct channel blockers. We conclude that the activity of Kir channels in the RPE is critically dependent on the regeneration of membrane PIP₂ by PI4 kinases and that this may explain the dependence of these channels on hydrolyzable ATP.

retinal pigment epithelium; Kir7.1; phosphatidylinositol 4-kinases; phosphatidylinositol 4,5-bisphosphate