Dynamics of PIP2 regulation and metabolism seen with PIP2‐sensitive ion channels and optical probes.

Abstract

KCNQ2/3 K+ channels require PIP2 to function. Activation of muscarinic M1 receptors (M1R) turns these channels off by Gq, PLC activation, and PIP2 depletion (Suh & Hille, 2002). To monitor steps of this signaling cascade and plasma membrane PIP2 dynamics, we use FRET pairs, translocation of probes, and the channel current in intact living tsA cells. M1Rs acting through PLC deplete PIP2, make DAG, and shut channels in ~10 s. Following FRET pairs in the receptor signaling pathway shows that receptor activation takes <0.1 s, receptor‐G‐protein interactions ~0.3 s, G protein activation ~2 s, and PLC is activated shortly after. Thus most of the delay in signaling to channels is the time it takes PLC to deplete PIP2 pools. Recovery of PIP2 after M1R agonist takes 100‐200 s. This recovery is wortmannin sensitive so PI(4)P needs to be regenerated as well as PIP2. Recovery time shortens with overexpression of PI 4‐kinase but not PIP 5‐kinase. PIP2 can also be depleted in a few seconds by activating a PIP2 5‐phosphatase with voltage steps (VSP) or with chemical dimerization, generating PI(4)P rather than IP3/DAG. Recovery from the perturbation by VSP takes only 15 s and becomes even faster if PIP 5‐kinase is overexpressed. It is not wortmannin sensitive. Thus the PIP 5‐kinase step in the synthesis of PIP2 is normally significantly faster than the PI 4‐kinase step. Supported by NIH grants RO1 NS08174, RO1 GM83913, T32 GM07108, and the HFSP.