Fluorescence changes reveal kinetic steps of muscarinic receptor-mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels

J Gen Physiol. 2009 Apr;133(4):347-59. doi: 10.1085/jgp.200810075.


G protein-coupled receptors initiate signaling cascades. M(1) muscarinic receptor (M(1)R) activation couples through Galpha(q) to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP(2)). Depletion of PIP(2) closes PIP(2)-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M(1)R activation, M(1)R/Gbeta interaction, Galpha(q)/Gbeta separation, Galpha(q)/PLC interaction, and PIP(2) hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M(1)R activation (<100 ms) and M(1)R/Gbeta interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Galpha(q)/Gbeta separation and Galpha(q)/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP(2) hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Galpha(q)/PLC interaction. Evidently, channel release of PIP(2) and closure are rapid, and the availability of active PLC limits the rate of M current suppression.

Publication types

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

MeSH terms

  • Animals
  • Cattle
  • Cells, Cultured
  • Humans
  • KCNQ2 Potassium Channel / genetics
  • KCNQ2 Potassium Channel / metabolism*
  • KCNQ3 Potassium Channel / genetics
  • KCNQ3 Potassium Channel / metabolism*
  • Mice
  • Microscopy, Fluorescence
  • Phosphatidylinositols / metabolism*
  • Phosphatidylinositols / pharmacokinetics
  • Photometry
  • Protein Binding / genetics
  • Rats
  • Receptors, Muscarinic / physiology*


  • KCNQ2 Potassium Channel
  • KCNQ2 protein, human
  • KCNQ3 Potassium Channel
  • Kcnq3 protein, rat
  • Phosphatidylinositols
  • Receptors, Muscarinic