The Journal of General Physiology
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Published online
doi:10.1085/jgp.200810075
The Journal of General Physiology, Vol. 133, No. 4, 347-359
The Rockefeller University Press, 0022-1295 $30.00
© Jensen et al.
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ARTICLE

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

Jill B. Jensen1, John S. Lyssand2, Chris Hague2, and Bertil Hille1

1 Department of Physiology and Biophysics, and 2 Department of Pharmacology, University of Washington School of Medicine, Seattle, WA 98195

Correspondence to Bertil Hille: hille{at}u.washington.edu

G protein–coupled receptors initiate signaling cascades. M1 muscarinic receptor (M1R) activation couples through G{alpha}q to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of PIP2 closes PIP2-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 M1R activation, M1R/Gβ interaction, G{alpha}q/Gβ separation, G{alpha}q/PLC interaction, and PIP2 hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M1R activation (<100 ms) and M1R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. G{alpha}q/Gβ separation and G{alpha}q/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP2 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 G{alpha}q/PLC interaction. Evidently, channel release of PIP2 and closure are rapid, and the availability of active PLC limits the rate of M current suppression.

Abbreviations used in this paper: ECFP, enhanced cyan fluorescent protein; EYFP; enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; GPCR, G protein–coupled receptor; GRK2, GPCR kinase 2; 3H-NMS, N-methyl-3H-scopolamine; IP3, inositol 1,4,5-trisphosphate; M1R, M1 muscarinic receptor; oxo-M, oxotremorine-methiodide; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; PLCβ, phospholipase C-β.

© 2009 Jensen et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).


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