Kinetics of PIP2 Metabolism and KCNQ2/3 Channel Regulation Studied With a Voltage-Sensitive Phosphatase in Living Cells

J Gen Physiol. 2010 Feb;135(2):99-114. doi: 10.1085/jgp.200910345.

Abstract

The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP(2)) is synthesized in two steps from phosphatidylinositol by lipid kinases. It then interacts with KCNQ channels and with pleckstrin homology (PH) domains among many other physiological protein targets. We measured and developed a quantitative description of these metabolic and protein interaction steps by perturbing the PIP(2) pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP(2) with a time constant of tau <300 ms and fully inhibits KCNQ currents in a similar time. PIP(2) was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, tau = 11 s. In contrast, resynthesis of PIP(2) after activation of phospholipase C by muscarinic receptors took approximately 130 s. These kinetic experiments showed that (1) PIP(2) activation of KCNQ channels obeys a cooperative square law, (2) the PIP(2) residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP(2) by PIP 5-kinase is fast and limited primarily by a step(s) that replenishes the pool of plasma membrane PI(4)P. We extend the kinetic model for signaling from M(1) muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910344), with this new information on PIP(2) synthesis and KCNQ interaction.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line
  • Electrophysiological Phenomena
  • Humans
  • KCNQ2 Potassium Channel / metabolism*
  • KCNQ2 Potassium Channel / physiology
  • KCNQ3 Potassium Channel / metabolism*
  • KCNQ3 Potassium Channel / physiology
  • Kinetics
  • Phosphatidylinositol 4,5-Diphosphate / metabolism*
  • Phosphoric Monoester Hydrolases / metabolism*
  • Phosphoric Monoester Hydrolases / physiology
  • Phosphotransferases (Alcohol Group Acceptor) / physiology
  • Protein Binding / physiology
  • Receptors, Muscarinic / metabolism
  • Receptors, Muscarinic / physiology
  • Signal Transduction / physiology

Substances

  • KCNQ2 Potassium Channel
  • KCNQ3 Potassium Channel
  • Phosphatidylinositol 4,5-Diphosphate
  • Receptors, Muscarinic
  • Phosphotransferases (Alcohol Group Acceptor)
  • 1-phosphatidylinositol-4-phosphate 5-kinase
  • Phosphoric Monoester Hydrolases