Capillaries form branching networks that surround all cells of the body. They allow oxygen and nutrient exchange between blood and tissue, but this is not their only role. Capillaries in the brain form a tight barrier that prevents components carried in the blood from easily reaching the brain compartment. They also detect the activity of neurons and trigger on-demand increases in blood flow to active regions of the brain. This role, revealed only recently, depends upon ion channels on the surface of the capillary cells. Active neurons release potassium ions, which open a type of ion channel called Kir2.1 that allows potassium inside the cell to flow out. This process is repeated in neighboring capillary cells until it reaches an upstream vessel, where it causes the vessel to relax and increase the blood flow.

Kir2.1 channels sit astride the membranes of capillary cells, where they can interact with other membrane molecules. One such molecule, called PIP₂, plays several roles in relaying signals from the outside to the inside of cells. It also physically interacts with channels in the membrane, including Kir2.1 channels. If PIP₂ levels are low, Kir2.1 channel activity decreases.

Here, Harraz et al. discovered that capillary cells contain another type of ion channel, called TRPV4, which is also regulated by PIP₂. But unlike Kir2.1, its activity increases when PIP₂ levels drop. Moreover, TRPV4 channels allow sodium and calcium ions to flow into the cell, which has an effect opposite to that of potassium flowing out of the cell.

Capillary cells also have receptor proteins called G_qPCRs that are activated by chemical signals released by active neurons in the brain. G_qPCRs break down PIP₂, so their activity turns Kir2.1 channels off and TRPV4 channels on. This resets the system so that it is ready to respond to new signals from active neurons. G_qPCRs work as molecular switches to control the balance between Kir2.1 and TRPV4 channels and turn brain blood flow up and down.

G_qPCRs and ion channels that depend on PIP₂ can also be found in other types of cells. These findings could reveal clues about how signals are switched on and off in different cells. Understanding the role of PIP₂ in signaling could also unveil what happens when signaling go wrong.

Endothelial cells (ECs) line the lumen of all blood vessels and are important regulators of artery and arteriole smooth muscle contractility. In capillaries, which lack an overlying smooth muscle cell layer, this regulatory function is absent. In these smallest of all blood vessels, ECs instead support the nutrient- and gas-exchange function characteristic of capillary beds generally. In the brain, capillary ECs (cECs) also constitute the blood-brain barrier, reflecting the presence of tight junctions between adjacent cECs. Not surprisingly given these differing cellular missions, the molecular repertoire of brain cECs and arterial/arteriolar ECs exhibit some marked differences. Notably, intermediate and small-conductance Ca²⁺-activated K⁺ channels (IK and SK, respectively), which are important in transducing elevations in intracellular Ca²⁺ concentration ([Ca²⁺]_i) into membrane potential hyperpolarization in artery/arteriolar ECs to cause relaxation of electrically coupled smooth muscle cells (Ledoux et al., 2008; Sonkusare et al., 2012; Taylor et al., 2003), are absent in brain cECs (Longden et al., 2017). However, signaling through G protein–coupled receptors of the Gα_q/11-subtype (G_qPCRs)—an important component of this smooth muscle regulatory axis—is robust in brain cECs (Harraz et al., 2018). In the canonical pathway of G_qPCR signaling, Gα_q released upon agonist binding activates phospholipase C (PLC), which hydrolyzes the minor inner leaflet phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP₂). Breakdown of PIP₂ yields diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃), the latter of which increases [Ca²⁺]_i by acting on its cognate receptor (IP₃R) on the endoplasmic reticulum (ER) to promote Ca²⁺ release from intracellular stores.

Blood delivery within the brain is mediated by an ever-narrowing vascular tree comprising surface arteries, penetrating (parenchymal) arterioles and a vast network of capillaries, which enormously extend the territory of perfusion (Blinder et al., 2013). Because the brain lacks substantial energy reserves, it relies on an on-demand mechanism for redistributing oxygen and nutrients to regions of higher neuronal activity, a process in which products released by active neurons trigger a local increase in blood flow. This use-dependent increase in local blood flow (functional hyperemia), mediated by a process termed neurovascular coupling (NVC), is essential for normal brain function (Iadecola and Nedergaard, 2007) and represents the physiological basis for functional magnetic resonance imaging (Raichle and Mintun, 2006).

We recently reported that brain capillaries act as a neuronal activity-sensing network, demonstrating that brain cECs are capable of initiating an electrical (hyperpolarizing) signal in response to neuronal activity that rapidly propagates upstream to dilate feeding parenchymal arterioles and increase blood flow locally at the site of signal initiation (Longden et al., 2017). We also established the molecular mechanism underlying this process, showing that extracellular K⁺—a byproduct of every neuronal action potential—is the critical mediator and identifying the Kir2.1 channel as the key molecular player (Longden et al., 2017). Kir2.1 channels are activated by external K⁺ and exhibit steep activation by hyperpolarization (Hibino et al., 2010; Longden and Nelson, 2015), characteristics that facilitate regenerative K⁺ conductance in neighboring cECs and enable long-range propagation of the hyperpolarizing signal (Longden et al., 2017). Intriguingly, we have since discovered that decreases in capillary endothelial PIP₂ levels induced by G_qPCRs agonists suppress Kir2.1 channel currents, reflecting the fact that PIP₂ is required for Kir2.1 channel activity (Harraz et al., 2018). Importantly, this suppressive effect of PIP₂ depletion exerts a major modulatory influence on K⁺-evoked hyperemic responses in vivo. In addition to regulating Kir2 family members, PIP₂ has been shown to bind to and regulate a diverse array of ion channels (Hille et al., 2015). Notable in this context, PIP₂ binding has been shown to inhibit several members of the transient receptor potential (TRP) family of non-selective cation channels, including the vanilloid subtype TRPV4 (Nilius et al., 2008; Rohacs, 2009; Takahashi et al., 2014), which we and others have shown is an important Ca²⁺ influx pathway in arterial and arteriolar ECs (Earley and Brayden, 2015; Sonkusare et al., 2014;Sonkusare et al., 2012; White et al., 2016).

The Kir2.1 channel appears to be the major K⁺ channel type in brain cECs (Longden et al., 2017). However, the identity of depolarizing (Na⁺/Ca²⁺-permeable) channels in cECs is not known. Here, we show that TRPV4 channels are present in cECs and exhibit an exceedingly low open probability under basal conditions due to tonic inhibition by PIP₂. We have further found that this inhibition is relieved through PIP₂ depletion, independent of the action of the PIP₂ hydrolysis products, DAG and IP₃. These findings provide a counterpoint to our recent demonstration that Kir2.1 channel activity is sustained by basal levels of PIP₂ and suppressed by G_qPCR-mediated PIP₂ depletion (Harraz et al., 2018). Collectively, our findings indicate that PIP₂ exerts opposite effects on TRPV4 and Kir2.1 channels in brain cECs, demonstrating that a single regulatory pathway governs the balance between two divergent signaling modalities—one depolarizing and the other hyperpolarizing—in the capillary endothelium.

We previously demonstrated that increases in [K⁺]_o associated with neuronal activity alter cerebral blood flow at the capillary level, showing that extracellular K⁺ activates capillary Kir2.1 channels, triggering a retrograde electrical (hyperpolarizing) signal that propagates upstream to dilate feeding arterioles and enhance blood flow to the active region (Longden et al., 2017). We have also shown that G_qPCR-mediated PIP₂ depletion inhibits capillary electrical signaling (Harraz et al., 2018), identifying a point of intersection between electrical signaling and endothelial G_qPCR activity. In the present study, we show that cECs express TRPV4 channels, and demonstrate that these channels are tonically suppressed by basal levels of PIP₂. Furthermore, we show that G_qPCR activation relieves TRPV4 channel inhibition through PIP₂ depletion. This paradigm introduces the phosphoinositide PIP₂ as a master regulator of TRPV4 and Kir2.1 signaling in the capillary endothelium and highlights the ability of G_qPCR activity to tune the balance of these signaling modalities to favor TRPV4 signaling (Figure 7).

The repertoire of functional ion channels in brain cECs remains incompletely characterized, although certain molecular features have recently come into focus. For instance, our evidence suggests that the inward rectifier Kir2.1 channel is the major K⁺ channel type in brain cECs (Longden et al., 2017), whereas Ca²⁺-sensitive SK and IK channels, which are present in arterial and arteriolar ECs and play a prominent role in regulating vascular tone (Ledoux et al., 2008; Sonkusare et al., 2012; Taylor et al., 2003), are not expressed in brain cECs (Longden et al., 2017). Notably, the identity of depolarizing (Na⁺ and/or Ca²⁺-permeable) channels in cECs, which we predict must be present to allow the membrane potential to reset to support repeated operation of our previously reported Kir2.1-dependent electrical signaling-based NVC mechanism, is not known. Our demonstration that the highly selective TRPV4 agonist, GSK101, induced currents in brain cECs that were eliminated by the TRPV4-specific antagonist, HC-067047, and were absent in cECs from TRPV4^-/- mice firmly establishes the presence of this non-selective cation channel in capillaries. In arterial and arteriolar ECs, TRPV4 channel-mediated Ca²⁺ influx is closely linked to activation of Ca²⁺-sensitive SK and IK channels and subsequent membrane potential hyperpolarization (Sonkusare et al., 2012). However, because cECs lack functional Ca²⁺-activated K⁺ channels (Longden et al., 2017), TRPV4-mediated influx of Na⁺/Ca²⁺ in these cells instead would lead to membrane potential depolarization (Behringer et al., 2017; Earley and Brayden, 2015). Collectively, these observations suggest that the TRPV4 channel is a major depolarizing element in cECs, although we cannot rule out the possibility that other, as yet unidentified, cation channels may contribute to membrane depolarization, as suggested by earlier studies (Csanády and Adam-Vizi, 2004; Popp and Gögelein, 1992; Csanády and Adam-Vizi, 2003). Our observations also highlight the fact that the functional role of TRPV4 channels is critically dependent on the expression and function of key associated proteins.

Intriguingly, and in striking contrast to the case in peripheral arterial ECs (Sonkusare et al., 2014; Sonkusare et al., 2012), the open probability of capillary TRPV4 channels in cECs was remarkably low under basal conditions and was increased by dialyzing out intracellular contents—the first link in the chain leading to our discovery that TRPV4 channels in cECs are intrinsically inhibited by intracellular ATP. It is well established that lipid kinases involved in PIP₂ synthesis require millimolar ATP for their activity (Balla and Balla, 2006; Hilgemann, 1997; Knight and Shokat, 2005; Suer et al., 2001), and it has been amply demonstrated that PIP₂ maintenance is dependent on ATP in multiple cell types (Suh and Hille, 2008; Ye et al., 2018; Zakharian et al., 2011), including cECs (Harraz et al., 2018). Three major lines of evidence presented here support the conclusion that cytosolic ATP-dependent maintenance of sustained, basal levels of PIP₂ suppresses TRPV4 channel activity. First, millimolar concentrations of hydrolyzable ATP suppressed capillary TRPV4 channel activity. Second, inclusion of PIP₂ analogs in the patch pipette inhibited TRPV4 currents. Third, scavenging PIP₂ or inhibiting its synthesis abrogated ATP-mediated inhibition. Notably, PIP₂ has been reported to directly interact with different residues on the TRPV4 N-terminus in heterologous expression systems (Garcia-Elias et al., 2013; Takahashi et al., 2014). However, these studies reached discrepant conclusions, with one suggesting that direct binding of PIP₂ to the ankyrin repeat domain of the TRPV4 channel inhibits different modes of TRPV4 activation (Takahashi et al., 2014) and the other reporting that PIP₂ is necessary for heat-, hypotonicity- or epoxyeicosatrienoic acid (EET)-induced channel activation by facilitating structural rearrangements of the channel (Garcia-Elias et al., 2013). These discrepancies may reflect divergent effects of PIP₂ on channel behavior through binding to multiple sites in the channel. Intriguingly, the putative endogenous TRPV4 channel activator (Earley and Brayden, 2015; Watanabe et al., 2003) 11,12-epoxyeicosatrienoic acid (11,12-EET, 1 µM) evoked currents in cECs even in the absence of dialyzed PIP₂ (Figure 3—figure supplement 2). In any case, our results are the first to report PIP₂-mediated suppression of TRPV4 channels in the endothelium and more broadly in native cells and are congruent with the results of Takahashi et al., 2014. Interestingly, a recent study provided structural evidence that lipid molecules are tightly bound to the selectivity filter of the TRPV4 channel pore, although the identity and function of these lipids were not characterized (Deng et al., 2018).

Canonical G_qPCR signaling involves PLC activation and subsequent PIP₂ breakdown into DAG and IP₃. Receptor agonists that activate G_qPCRs can dramatically lower PIP₂ by promoting its hydrolysis at rates that exceed those of PIP₂ re-synthesis. We show here that PGE₂, which has previously been postulated to act as an NVC agent through actions on G_s-coupled EP₂/EP₄ receptors in arteriolar smooth muscle (Lacroix et al., 2015; Zonta et al., 2003), activates TRPV4 channels in cECs independent of the action of the PIP₂ metabolites, DAG and IP₃, by relieving PIP₂-mediated suppression (Figure 4, Figure 5). In contrast, we recently showed that G_qPCR agonists exert the opposite effect on capillary Kir2.1 channels, inhibiting their ability to mediate capillary-to-arteriole electrical signaling (Harraz et al., 2018). In keeping with these previous findings and our current observations, simultaneous monitoring of TRPV4 and Kir2.1 channel currents confirmed the bidirectional effects of G_qPCR agonists on the two channels (Figure 6). This two-way modulation (Figure 7) is unique as it indicates that a single G_qPCR signaling cascade is capable of altering the balance between electrical Kir2.1 signaling (inhibition) and TRPV4 signaling (facilitation). Given the absence of Ca²⁺-activated K⁺ channels in cECs, noted above, G_qPCR signaling-induced PIP₂ depletion would likely depolarize cECs through the simultaneous disinhibition of depolarizing TRPV4 channels (this study) and deactivation of hyperpolarizing Kir2.1 channels (Harraz et al., 2018).

Based on our direct Kir2.1 current measurements (Harraz et al., 2018; Longden et al., 2017) and the known voltage- and K⁺-dependence of Kir2.1 channels (Longden and Nelson, 2015), we estimate that the outward current through these channels at physiological membrane potentials (about −40 mV) and external K⁺ (3 mM) is ~6 fA. Elevation of external K⁺ to 10 mM would increase Kir2 current at this voltage to ~260 fA. Though seemingly miniscule, such small membrane currents are precisely what is needed to ensure conduction fidelity. We have measured a sustained and profound hyperpolarization of about −25 mV in arteriolar smooth muscle in response to capillary stimulation with 10 mM K⁺ (Longden et al., 2017). Our computational modeling indicates that a stable membrane hyperpolarization of 25 mV requires the outward (hyperpolarizing) current to greatly exceed the inward (depolarizing) current. Our estimate of basal, PIP₂-suppressed TRPV4 current at −40 mV is about −80 fA. G_qPCR activation increases open probability ~6 fold, producing a current (400–500 fA) sufficient to effectively short circuit K⁺-induced hyperpolarization and cripple this key Kir2.1-based NVC mechanism. In conclusion, the low level of TRPV4 channel activity aligns with the functional role of capillaries.

Activation of capillary G_qPCRs should also trigger Ca²⁺ signals in cECs, presumably through IP₃-mediated Ca²⁺ release from intracellular stores as well as disinhibition of TRPV4 channels (as described here) and subsequent Ca²⁺ influx. Such capillary Ca²⁺ signals should positively influence hemodynamics—and thus functional hyperemia—providing a plausible explanation for the role of G_qPCR agonists in the NVC process, presumably through the Ca²⁺-dependent activation of nitric oxide synthase (NOS) and subsequent release of the vasodilator, NO (Förstermann et al., 1991; Marziano et al., 2017). Capillary Ca²⁺ signaling might also serve an entirely different purpose in functional hyperemia: because local Ca²⁺ signals represent sites of PIP₂ depletion (through G_qPCR signaling), and thus membrane potential depolarization (through TRPV4 disinhibition and Kir2.1 deactivation), they are likely to interfere with the progression of electrical signals generated further down the vascular tree. Accordingly, these signals may represent ‘stop signs’ or ‘speed bumps’ that play a role in redirecting hyperpolarizing (vasodilatory) signals away from certain brain areas, in addition to their role in resetting the capillary membrane potential, noted above.

Given the role of PIP₂ in negatively regulating TRPV4 channels, the exceedingly low basal TRPV4 activity and diminished sensitivity to GSK101 in cECs (Figure 1) compared with mesenteric artery ECs (Sonkusare et al., 2012) suggest differences in the PIP₂ ‘set point’ in these two vascular beds. This supposition has important physiological implications for electrical signaling in the brain. Our data suggest that PIP₂ levels in brain cECs are sufficient to saturate Kir2.1 channels (Harraz et al., 2018) and tune TRPV4 channel currents to levels that prevent inappropriate depolarization of the membrane potential (Figure 3). In the absence of PIP₂ suppression of TRPV4 channel-mediated inward currents, it is unlikely that increased outward K⁺ currents through Kir2.1 channels in response to elevated K⁺ would be sufficient to cause membrane potential hyperpolarization. The mechanistic basis for the apparently higher PIP₂ set point in brain cECs is currently unknown, but could include lower constitutive G_qPCR activity, lower microenvironmental levels of G_qPCR agonists and/or decreased G_qPCR expression—and thus diminished PIP₂ breakdown. Along these lines, a recent single-cell transcriptomics analysis showed that Gα_q (Gnaq) transcript levels in the mouse brain endothelium are reduced compared with those in peripheral pulmonary ECs (Vanlandewijck et al., 2018). Alternatively, differences in the set point could be indicative of more robust PIP₂ synthesis in brain capillaries, a speculation that is supported by the higher mitochondrial content—and hence ATP synthesis—in highly active brain cECs compared with other ECs (Oldendorf and Brown, 1975; Oldendorf et al., 1977).

In conclusion, this study provides compelling evidence that brain capillaries express TRPV4 channels and further shows that the activity of these channels is physiologically suppressed by basal levels of PIP₂. This introduces PIP₂ and its modulation by G_qPCR agonists as major regulators of brain capillary signaling. When maintained at sufficient levels, PIP₂ inhibits TRPV4 channels and supports capillary-to-arteriole electrical signaling, but in response to G_qPCR activation, PIP₂ levels are reduced, enhancing TRPV4 signaling and inhibiting retrograde electrical signaling via Kir2.1 channels. Capillary G_qPCRs can therefore be envisioned as molecular switches that dictate the signaling modality in the brain microvasculature.

All data generated or analyzed during this study are included in the manuscript and supporting source data files.

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Author details

1. Osama F Harraz

   Department of Pharmacology, University of Vermont, Burlington, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2061-1100

2. Thomas A Longden

   Department of Pharmacology, University of Vermont, Burlington, United States

   Contribution

   Funding acquisition, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. David Hill-Eubanks

   Department of Pharmacology, University of Vermont, Burlington, United States

   Contribution

   Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

4. Mark T Nelson

   1. Department of Pharmacology, University of Vermont, Burlington, United States
   2. Institute of Cardiovascular Sciences, Manchester, United Kingdom

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

   For correspondence

   Mark.Nelson@uvm.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6608-8784

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