Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP2 depletion

Abstract

Brain capillaries play a critical role in sensing neural activity and translating it into dynamic changes in cerebral blood flow to serve the metabolic needs of the brain. The molecular cornerstone of this mechanism is the capillary endothelial cell inward rectifier K⁺ (Kir2.1) channel, which is activated by neuronal activity-dependent increases in external K⁺ concentration, producing a propagating hyperpolarizing electrical signal that dilates upstream arterioles. Here, we identify a key regulator of this process, demonstrating that phosphatidylinositol 4,5-bisphosphate (PIP₂) is an intrinsic modulator of capillary Kir2.1-mediated signaling. We further show that PIP₂ depletion through activation of G_q protein-coupled receptors (G_qPCRs) cripples capillary-to-arteriole signal transduction in vitro and in vivo, highlighting the potential regulatory linkage between G_qPCR-dependent and electrical neurovascular-coupling mechanisms. These results collectively show that PIP₂ sets the gain of capillary-initiated electrical signaling by modulating Kir2.1 channels. Endothelial PIP₂ levels would therefore shape the extent of retrograde signaling and modulate cerebral blood flow.

Keywords: GPCR; PIP2; cerebral blood flow; neurovascular coupling; potassium channels.

Fig. 1.

Kir2.1 activity in capillary endothelial cells is sustained by an ATP-dependent mechanism. (A) Representative traces of Kir2.1 currents in freshly isolated capillary endothelial cells (cECs) bathed in 60 mM K⁺, recorded from 0 to 20 or 25 min using voltage-ramps (−140 to 40 mV). (A, Left) Kir2.1 currents recorded in the conventional whole-cell configuration (dialyzed cytoplasm, 0 mM Mg-ATP in the pipette solution). (A, Middle) Kir2.1 currents recorded in the perforated whole-cell configuration (intact cytoplasm). (A, Right) Kir2.1 currents recorded in the conventional whole-cell configuration in a cEC dialyzed with 1 mM Mg-ATP. (B) Summary data showing normalized Kir2.1 currents over time, recorded at −140 mV in the conventional whole-cell configuration (dialyzed cytoplasm) with 0 mM Mg-ATP in the pipette solution (black line), in the perforated whole-cell configuration (intact cytoplasm; gray line), and in the conventional whole-cell configuration (dialyzed cytoplasm) with 1 mM Mg-ATP in the pipette solution (red line). Error bars represent SEM (n = 6–9 per condition). (C) Summary data showing the concentration dependence and hydrolysis requirement for Mg-ATP–mediated Kir2.1 current preservation (duration, 15 min). Values are presented as means ± SEM (*P < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test; n = 5–9 for Mg-ATP experiments and n = 4 for ATP-γ-S experiments). %I/I_max is Kir2.1 current normalized to the maximum current (at t₀) and expressed as a percentage. n.s., not significant.

Fig. 2.

Intracellular ATP and PIP₂ maintain Kir2.1 currents. (A) Schematic diagram showing the ATP-dependent synthesis steps and pharmacological interventions in the pathway leading to the production of PIP₂. (B) Representative traces of Kir2.1 currents recorded over 25 min in the conventional whole-cell configuration in a capillary endothelial cell (cEC) dialyzed with a pipette solution containing 0 mM Mg-ATP, with 10 µM diC8-PIP₂. (C) Changes in Kir2.1 currents over time, recorded in the conventional whole-cell configuration in cECs dialyzed with a pipette solution containing 0 mM Mg-ATP, with or without (control) 10 µM diC8-PIP₂. Currents obtained at 15 min are expressed as a percentage relative to those at t₀ (time of acquisition of whole-cell electrical access). Data are presented as means ± SEM (**P < 0.01 unpaired Student’s t test, n = 9–10). (D) Individual-value plots of peak inward currents in cECs, measured at −140 mV (at t₀) using the perforated whole-cell configuration (intact cytoplasm; gray) or conventional whole-cell configuration in cECs dialyzed with a pipette solution containing 0 mM Mg-ATP (black), 1 mM Mg-ATP (red), or 0 mM Mg-ATP + 10 µM diC8-PIP₂ (blue). Whole-cell capacitance averaged 8.6 pF. There were no significant differences among groups (one-way ANOVA followed by Dunnett’s multiple comparisons test, n = 19–57). (E) Representative traces of Kir2.1 currents in a cEC with intact cytoplasm (perforated configuration) before (control) and 15 min after incubation with the PIP5K inhibitor UNC3230 (100 nM). (F) Individual-value plots showing effects of the PIP₂ synthesis inhibitors PIK93 (PI4K inhibitor, 300 nM), PAO (PI4K inhibitor, 10 µM), and UNC3230 (PIP5K inhibitor, 100 nM) on Kir2.1 currents in cytoplasm-intact cECs. Inhibitors were bath-applied immediately after t₀, and currents were compared before and 15 min after incubation (*P < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test).

Fig. 3.

PGE₂ inhibits Kir2.1 current in cECs by reducing PIP₂ levels. (A) Schematic depiction of PIP₂ depletion by G_qPCR activation through PLC-mediated hydrolysis to IP₃ and diacylglycerol (DAG). (B) Representative traces of Kir2.1 currents in a dialyzed capillary endothelial cell (cEC; 0 mM Mg-ATP) at different time points after addition of PGE₂ (2 µM) showing accelerated current decline following G_qPCR activation. (C) Individual-value plots showing the enhancement of cEC Kir2.1 current decline by bath-applied PGE₂ (2 µM; n = 5) compared with time controls (no PGE₂; n = 9; ^#P < 0.05 unpaired Student’s t test) and rescue by 10 µM diC8-PIP₂ (dialyzed; n = 3) or 10 µM U73122 (bath-applied; n = 3). Currents were recorded upon access to the cell interior (t₀) and after 15 min in cECs dialyzed with 0 mM Mg-ATP-pipette solution. Changes in Kir2.1 currents were calculated as values obtained at 15 min relative to those at t₀, expressed as a percentage. Individual data points are shown together with means (long horizontal lines) and SEM (error bars) (**P < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test). (D) Representative current traces showing no effect of the PKC inhibitor Gö6976 (1 µM; bath-applied) or rapid cytosolic Ca²⁺ chelation with BAPTA (5.4 mM; dialyzed) on the PGE₂-induced decline of Kir2.1 currents in cECs dialyzed with 0 mM Mg-ATP. (E) Individual-value plots showing the effects of the prostanoid receptor blockers AH6809 (10 µM, n = 3) and SC51322 (1 µM, n = 3) on the enhancement of Kir2.1 current decline in cECs by PGE₂, recorded under cytoplasm-intact conditions over a 15-min period. Changes in Kir2.1 currents were calculated as values obtained at 15 min relative to those at t₀, expressed as a percentage. Individual data points are shown together with means (long horizontal lines) and SEM (error bars) (^####P < 0.0001, unpaired Student’s t test, n = 6; **P < 0.01, one-way ANOVA followed by Dunnett’s multiple comparisons test). (F) Effects of G_qPCR agonists on normalized Kir2.1 current decline in cECs. Kir2.1 currents were recorded in the perforated patch configuration over 15 min in the absence (control) or presence of bath-applied PGE₂ (2 µM), carbachol (CCh, 10 µM), oxotremorine M (Oxo-M, 10 µM), SLIGRL-NH₂ (5 µM), or ATP (30 µM). Red horizontal lines indicate means (n = 4–6 each).

Fig. 4.

G_qPCR stimulation cripples capillary-to-arteriole electrical signaling. (A) Representative diameter recording showing the time course of the inhibitory effect of bath-applied PGE₂ (1 µM) on upstream arteriolar dilations induced by successive focal applications of 10 mM K⁺ (18 s, 5 psi) onto capillary segments in a capillary-parenchymal arteriole (CaPA) preparation (schematic, Right Inset). Relations above the trace indicate the processes occurring in the presence of PGE₂ [dissociation of PIP₂ from Kir2.1 and hydrolysis of PIP₂ to diacylglycerol (DAG)] and washout (reassociation of PIP₂ with Kir2.1). (B) Summary data for experiment in A, showing K⁺-induced dilations from five CaPA preparations (n = 5 mice), calculated as a percentage of maximal diameter responses (obtained in 0 mM Ca²⁺ at the conclusion of each experiment). Results were best fit as a plateau (lag phase) followed by one-phase exponential decay (R² = 0.85). Lag phase (X₀) ≈ 18 min; time constant of the postplateau exponential decay phase (τ_decay) ≈ 4 min. (C) Kir2.1 current decline following application of 2 µM PGE₂ onto capillary endothelial cells (cECs) at t₀ (i.e., upon achieving electrical access), recorded in the perforated-patch (intact cytoplasm) configuration. Time constant of the exponential decay phase (τ_decay) ≈ 12 min (one-phase exponential decay, R² = 0.85). Note the absence of a lag phase for Kir2.1 current decline. At X₀ (18 min), corresponding to the lag phase before detecting a decrease in dilatory response (in B), Kir2.1 current had declined by ∼53%.

Fig. 5.

Activation of cEC muscarinic receptors attenuates K⁺-induced increases in capillary RBC flux in vivo. (A) A 3D projection depicting the positioning of a pipette containing artificial cerebrospinal fluid with 10 mM K⁺ and TRITC-dextran (red) adjacent to a brain cortex capillary in vivo. Green, FITC-dextran circulating in blood plasma. (B, Top) Raw capillary line-scan data showing RBCs (black streaks) in plasma (green); the x axis is time and the y axis is scanned capillary distance (d). (B, Middle and Bottom) Line scans at baseline and in response to ejection of K⁺ (10 mM) onto the target capillary in a control (saline-injected) mouse and a mouse injected with carbachol (CCh, 0.6 µg/kg). Mice were systemically administered saline or CCh 20 min before applying 10 mM K⁺ by pressure ejection. At the conclusion of experiments, 0 mM Ca²⁺/200 µM diltiazem was applied to the brain surface to evoke near-maximal arteriolar dilation and increase blood flow to the capillary bed to provide a frame of reference for the modest and submaximal increases in basal RBC flux sometimes observed in CCh-injected mice. Each line scan spans 1 s. (C) Time course of capillary RBC flux corresponding to the experiments in B in response to ejection of K⁺ (10 mM) onto a capillary in a control (saline-injected) and a CCh-treated mouse, showing elimination of K⁺-induced dilation by activation of capillary endothelial cell muscarinic receptors. (D) Changes in K⁺ (10 mM)-induced capillary RBC flux over 30 min in saline- and CCh-treated mice (n = 6–7). Changes in flux at 10, 20, and 30 min were normalized to their respective baseline values. (E) Summary data showing the percentage change in RBC flux in response to K⁺ (10 mM) 20 min after saline (n = 5) or CCh (n = 7) treatment (**P < 0.01, unpaired Student’s t test).