Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

Abstract

The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) 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 PIP2 pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP2 with a time constant of τ <300 ms and fully inhibits KCNQ currents in a similar time. PIP2 was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, τ = 11 s. In contrast, resynthesis of PIP2 after activation of phospholipase C by muscarinic receptors took ∼130 s. These kinetic experiments showed that (1) PIP2 activation of KCNQ channels obeys a cooperative square law, (2) the PIP2 residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP2 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 M1 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 PIP2 synthesis and KCNQ interaction.