Purinergic inhibition of the epithelial Na + transport via hydrolysis of PIP 2

Stimulation of purinergic receptors inhibits amiloride‐sensitive Na + transport in epithelial tissues by an unknown mechanism. Because previous studies excluded the role of intracellular Ca 2+ or protein kinase C, we examined whether purinergic regulation of Na + absorption occurs via hydrolysis of phospholipid such as phosphatidylinositol‐bisphosphates (PIP 2 ). Inhibition of amiloride‐sensitive short‐circuit currents (I sc‐Amil ) by adenine 5′‐triphosphate (ATP) in native tracheal epithelia and M1 collecting duct cells was suppressed by binding neomycin to PIP 2 , and recovery from ATP inhibition was abolished by blocking phosphatidylinositol‐4‐kinase or diacylglycerol kinase. Stimulation by ATP depleted PIP 2 from apical membranes, and PIP 2 co‐ immunoprecipitated the β subunit of ENaC. ENaC was inhibited by ATP stimulation of P2Y 2 receptors in Xenopus oocytes. Mutations in the PIP 2 binding domain of βENaC but not γENaC reduced ENaC currents without affecting surface expression. Collectively, these data supply evidence for a novel and physiologically relevant regulation of ENaC in epithelial tissues. Although surface expression is controlled by its C terminus, N‐terminal binding of βENaC to PIP 2 determines channel activity.