Nucleotides and phospholipids compete for binding to the C terminus of K(ATP) channels

Inwardly rectifying, ATP-sensitive K(+) channels (K(ATP)) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P(2), antagonize nucleotide inhibition of K(ATP) channels enhancing the coupling of metabolic events to cell electrical or transport activity. The mechanism by which phospholipids relieve ATP block is unclear. We have shown that maltose-binding fusion proteins (MBP) containing the COOH termini of K(ATP) channels (Kir1.1, Kir6.1, and Kir6.2) form functional tetramers that directly bind at least two ATP molecules with negative cooperativity. Here we show that purified phosphatidylinositol phospholipids compete for 2,4,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of K(ATP) channels with EC(50) values for PIP(2) between 6–8 μM. The phospholipid potency profile was PIP(3) > PIP(2) = PIP > PI, suggesting that net phospholipid charge was important. A role for head group charge was supported by polycations (neomycin, spermine, and polylysine) reversing the effect of PIP(2) on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP(2), inositol(1,4,5)P(3) (IP(3)), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP(2) was also necessary for its effect on TNP-ATP binding. Indeed, neutral and charged lipids had weak, but significant, effects on TNP-ATP binding. Whereas μM concentrations of PIP(2) could compete with TNP-ATP, we found that mM concentrations of MgATP were required to compete with PIP(2) for binding to these K(ATP) channel COOH termini. Thus the COOH termini of K(ATP) channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.