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

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.

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 CFP_C fluorescence (blue trace, left axis) and YFP_C fluorescence (yellow trace, right axis), and the bottom panel shows the corrected ratio, YFP_C/CFP_C (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 τ_off = 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 CFP_C fluorescence (blue trace, left axis) and YFP_C fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFP_C/CFP_C (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 Gα_q/Gβ₁ separation. All cells coexpress M₁R, Gα_q-CFP, Gβ₁-YFP, Gγ₂, and GRK2, except GRK2 is absent in one part of D. (A) Cartoon of Gα_q-CFP and Gβ₁-YFP constructs and cognate G proteins. (B) Confocal images of a group of cells expressing Gα_q-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 CFP_C fluorescence (blue trace, left axis) and YFP_C fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFP_C/CFP_C (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 Gα_q/PLCβ₁ interaction. (A) Cartoon of Gα_q-CFP, PLCβ₁-YFP, and cognate G proteins. (B) Confocal images of a pair of cells coexpressing Gα_q-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 CFP_C fluorescence (blue trace, left axis) and YFP_C fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFP_C/CFP_C (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 CFP_C fluorescence (blue trace, left axis) and YFP_C fluorescence (yellow trace, right axis), and the bottom panel shows the ratio, YFP_C/CFP_C (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.