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

Comparative Study

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

Jill B Jensen et al. J Gen Physiol. 2009 Apr.

Abstract

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.

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Figures

Figure 1.

Kinetics of M1R activation. (A) Cartoon of the double-labeled M1R construct, M1R-YFP-CFP. (B) Confocal images of three resting cells, only one of which expresses the M1R. The transfected cell shows the distributions of cyan and yellow fluorescence. Bar, 10 µm. (C) FRETr photometry time course for a single cell. The top panel shows corrected CFPC fluorescence (blue trace, left axis) and YFPC fluorescence (yellow trace, right axis), and the bottom panel shows the corrected ratio, YFPC/CFPC (black), for a 5-s exposure to 10 µM oxo-M. Sampling frequency: 1 Hz during baseline and 10 Hz during agonist. (D) Normalized mean time course for five 5-s exposures to oxo-M in a single cell (same cell as C). Black line is a single-exponential fit with τoff = 180 ms.

Figure 2.

Kinetics of M1R/Gβ1 interaction. (A) Cartoon of M1R-CFP and Gβ1-YFP constructs and cognate G proteins. (B) Confocal images of a pair of cells expressing M1R-CFP and Gβ1-YFP. Bar, 10 µm. (C) FRETr photometry time course for a single cell undergoing a 5-s exposure to 10 µM oxo-M. The top panel shows CFPC fluorescence (blue trace, left axis) and YFPC fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFPC/CFPC (black). Sampling frequency: 2 Hz during baseline and 20 Hz during agonist. (D) Mean time course for 5-s exposures to oxo-M in six cells. Note the different time scales for onset and washout. Mean ± SEM (E) FRETr time course for a single cell. Oxo-M was stepped to different concentrations ranging from 10 nM to 50 µM as labeled. (F) FRETr concentration–response curve from steady-state values in E for six cells. Mean ± SEM.

Figure 3.

Kinetics of Gαq/Gβ1 separation. All cells coexpress M1R, Gαq-CFP, Gβ1-YFP, Gγ2, and GRK2, except GRK2 is absent in one part of D. (A) Cartoon of Gαq-CFP and Gβ1-YFP constructs and cognate G proteins. (B) Confocal images of a group of cells expressing Gαq-CFP and Gβ1-YFP in the presence of GRK2. Bar, 10 µm. (C) FRETr photometry time course for a single cell undergoing a 10-s exposure to 10 µM oxo-M. The top panel shows CFPC fluorescence (blue trace, left axis) and YFPC fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFPC/CFPC (black). Sampling frequency: 2 Hz during baseline and 10 Hz during agonist. (D) Mean time course for 10-s exposures to oxo-M in 10 cells in the absence (open circles) and 8 cells in the presence (closed circles) of GRK2. Note the different time scales for onset and washout. Mean ± SEM. For clarity in display, points were pooled in 500-ms bins for onset and 4-s bins for washout. (E) FRETr time course for a single cell. Oxo-M was stepped to different concentrations ranging from 1 nM to 10 µM as labeled. For clarity in display, trace is smoothed. (F) FRETr concentration–response curve from steady-state values in E for six cells. Mean ± SEM.

Figure 4.

Kinetics of Gαq/PLCβ1 interaction. (A) Cartoon of Gαq-CFP, PLCβ1-YFP, and cognate G proteins. (B) Confocal images of a pair of cells coexpressing Gαq-CFP and PLCβ1-YFP. Bar, 10 µm. (C) FRETr photometry time course for a single cell undergoing a 5-s exposure to 10 µM oxo-M. The top panel shows CFPC fluorescence (blue trace, left axis) and YFPC fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFPC/CFPC (black). Sampling frequency: 2 Hz during baseline and 20 Hz during agonist. (D) Mean time course for 5-s exposures to oxo-M in 10 cells. Mean ± SEM. Note the different time scales for onset and washout. For clarity in display, points from fast sampling were pooled in 200-ms bins. (E) FRETr concentration–response time course for a single cell. Oxo-M from 10 nM to 50 µM as labeled. For clarity in display, trace is smoothed. (F) FRETr concentration–response curve from steady-state values in E for four cells. Mean ± SEM.

Figure 5.

Kinetics of PIP2 hydrolysis. (A) Cartoon of PH(PLCδ1)-CFP, PH(PLCδ1)-YFP, Kv7.2/7.3 channels, and PIP2. PLC hydrolyzes PIP2 to send PH probes to the cytosol. (B) Confocal images of three cells expressing PH-CFP and PH-YFP. Bar, 10 µm. (C) FRETr photometry time course for a single cell undergoing a 20-s exposure to 10 mM oxo-M. The top panel shows CFPC fluorescence (blue trace, left axis) and YFPC fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFPC/CFPC (black). Sampling frequency: 2 Hz throughout. (D) Mean time course for 20-s exposures to oxo-M in 22 cells. Mean ± SEM. Note the different time scales for onset and washout. For clarity in display, points were pooled in 1-s bins for onset and 10-s bins for washout. (E) FRETr concentration–response time course for a single cell. Oxo-M from 1 nM to 10 µM as labeled. (F) FRETr concentration–response curve from steady-state values in E for 12 cells. Mean ± SEM.

Figure 6.

Kinetics of Kv7.2/7.3 channel closure. (A) Time course for current from a single voltage–clamped cell undergoing a 20-s exposure to 10 µM oxo-M. Current is steady-state measured at −20 mV. Sampling frequency: 0.25 Hz during baseline and 200 Hz during agonist. (B) Individual current traces corresponding to points in A. Voltage was stepped from −20 to −60 mV for 500 ms every 4 s. (C) Time course for normalized mean current in five cells. Note the different time scales for onset and washout. For clarity in display, points from fast sampling were pooled in 1-s bins and points from slow sampling were pooled in 20-s bins. (D) M current concentration–response time course for a single cell. Oxo-M from 1 nM to 10 μM as labeled. (E) M current concentration–response curve from steady-state values for eight cells. Mean ± SEM.

Figure 7.

PLC speeds and PH probes slow M current suppression. (A and B) Normalized mean M current at −20 mV from six cells expressing either transfected PLCβ1-YFP (open circles, A) or low levels of PH(PLCδ1)-CFP and PH(PLCδ1)-YFP (open circles, B). For comparison, control M current from five cells lacking exogenous PLC or PH probes (black circles). Note the different time scales for onset and washout.

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