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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_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_q/Gβ separation, G_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_q/Gβ separation and G_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_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.

© 2009 Jensen et al.
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