Figure 5. PGE₂ relieves PIP₂-mediated TRPV4 channel suppression.

(A) Representative current-voltage plots obtained from a cEC dialyzed with 1 mM Mg-ATP and treated consecutively with GSK101 (100 nM) and RuR (1 µM) followed by 2 µM PGE₂. (B) Summary individual-value plot of GSK101-induced TRPV4 currents at 100 mV in cECs dialyzed with 1 mM Mg-ATP in the absence (black; n = 27) and presence (orange; n = 15) of 2 µM PGE₂. Incubation of cECs with PGE₂ lasted ~15 min. Data in B are means (column bars) ± SEM (error bars, ****p<0.0001, unpaired Student’s t-test). (C) Top: Schematic diagram showing the G_qPCR-dependent hydrolysis of PIP₂ and the interventions used to test different components of the proposed pathway. Bottom: Summary data showing GSK101 (100 nM)-induced currents recorded at 100 mV in cECs dialyzed with 1 mM Mg-ATP. Currents were recorded in the absence and presence of 2 µM PGE₂ (orange shading), with or without (control) the indicated interventions. Concentrations (and application method): HC-067047, 1 µM (bath); diC8-PIP₂, 10 µM (pipette), U73122, 10 µM (bath); U73343, 10 µM (bath); AH6809, 10 µM (bath); SC51322, 1 µM (bath); calphostin C, 0.5 µM (bath); Gö6976, 1 µM (bath); CPA, 30 µM (bath); BAPTA, 5.4 mM (pipette). For bath application, pharmacological agents were added 10–15 min before PGE₂ application.

Figure 5—figure supplement 1. PGE₂ relieves TRPV4 current inhibition.

(A) Current-voltage relationship recorded in a cEC dialyzed with 1 mM Mg-ATP using voltage ramps (−100 to 100 mV). The circled numbers indicated the sequential and cumulative application of GSK101 (100 nM)+RuR (1 µM) followed by 2 µM PGE₂ and lastly, the TRPV4 blocker HC-067047 (1 µM). (B) Representative scatter plots of peak current amplitudes (at 100 mV) in three cECs dialyzed with 1 mM Mg-ATP and treated with 100 nM GSK101 followed by 2 µM PGE₂ (or PGE₂ first followed by GSK101). Note that PGE₂-induced activation of TRPV4 current plateaued after ~13–15 min. (C) Scatter plots of normalized GSK101-induced outward currents (at 100 mV), normalized to the current at zero time, I₀ (when 2 µM PGE₂ was applied), in two cECs, one dialyzed with 1 mM Mg-ATP (orange symbols) and the other dialyzed with 1 mM Mg-ATP +10 µM diC8-PIP₂ (black symbols). Currents normalized to I₀ are presented before and after the application of PGE₂ for 35 min.

Figure 5—figure supplement 2. Different G_qPCR agonists differentially regulate TRPV4 activity.

Summary data showing TRPV4 currents evoked by 100 nM GSK101 at 100 mV in Mg-ATP (1 mM)-dialyzed cECs. Cells were treated with GSK101 alone (no agonist) or together with the G_qPCR agonist PGE₂ (2 µM), carbachol (CCh; 10 µM), SLIGRL (5 µM), or Na-ATP (50 µM). cECs were incubated with different agonists for 15 min while TRPV4 current development was monitored. Data are presented as means ± SEM (**p<0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test; n = 11–17 cECs).