Nucleotides and phospholipids compete for binding to the C terminus of KATP channels

Gordon G. MacGregor, Ke Dong, Carlos G. Vanoye, Lieqi Tang, Gerhard Giebisch, Steven C. Hebert*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

106 Scopus citations

Abstract

Inwardly rectifying, ATP-sensitive K+ channels (KATP) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P2, antagonize nucleotide inhibition of KATP 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 KATP 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 KATP channels with EC50 values for PIP2 between 6-8 μM. The phospholipid potency profile was PIP3 > PIP2 = 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 PIP2 on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP2, inositol(1,4,5)P3 (IP3), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP2 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 PIP2 could compete with TNP-ATP, we found that μM concentrations of MgATP were required to compete with PIP2 for binding to these KATP channel COOH termini. Thus the COOH termini of KATP channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.

Original languageEnglish (US)
Pages (from-to)2726-2731
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number5
DOIs
StatePublished - Mar 5 2002

ASJC Scopus subject areas

  • General

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