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

Journal of General Physiology, ISSN: 0022-1295, Vol: 133, Issue: 4, Page: 347-359
2009
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G protein-coupled receptors initiate signaling cascades. M 1 muscarinic receptor (M 1 R) activation couples through G α 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 fl uorescence resonance energy transfer for M 1 R activation, M 1 R/Gβ interaction, G α q /G β separation, G α /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/G β interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. G α /G β separation and G α /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 G α /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. © 2009 Jensen et al.

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