Roles of transient receptor potential channels in regulation of vascular and epithelial barriers

ABSTRACT

Transient receptor potential (TRP) channels are a ubiquitously expressed multi-family group of cation channels that are critical to signaling events in many tissues. Their roles have been documented in many physiologic and pathologic conditions. Nevertheless, direct studies of their roles in maintain barrier function in endothelial and epithelia are relatively infrequent. This seems somewhat surprising considering that calcium ion concentrations are known to regulate barrier function. This short review provides an introduction to TRP channels and reviews some of the work in which investigators directly studied the role of TRP channels in endothelial permeability to electric current, solute, or leukocytes during the inflammatory response.

The mammalian transient receptor potential (TRP) cation channels are a group of 28 (in humans) structurally related transmembrane proteins that are grouped into 6 families based on amino acid sequence.¹ These channels are ubiquitously expressed throughout the body, with representative members of all or most families expressed in most cells. TRP channels are activated by a variety of signals and are non-selectively permeable to calcium (Ca²⁺), magnesium (Mg²⁺), and sodium (Na⁺). However, TRPC channels, for example, are more permeable to Ca²⁺ than the other cations. Since Ca²⁺ is such a common second messenger and is critical for cell adhesion and monolayer stability, one would imagine that there is a vast literature on the role of TRP channels in regulation of barrier function. In fact, although these channels are well-studied, a great deal remains to be learned about them, particularly regarding their regulation of endothelial and epithelial barriers. At the time of this writing, a search of PubMed for “transient receptor potential channels” turns up over 12,500 results of which 2,150 are reviews. There is a vast store of knowledge about these receptors and how they are activated and regulated; and how they function in a wide variety of metabolic and signaling processes at the cellular and tissue level. However, a search for “transient receptor potential channels AND barrier” turns up only 191, of which 60 are reviews, and most do not address the function of these channels in maintaining barrier function. Therefore, this mini-mini review will discuss what is known on this subject. There is still a vast amount that we still need to learn.

TRP channels consist of homotetramers (and in some cases heterotetramers) of subunits that have a related structure (See Fig. 1). They have 6 membrane-spanning domains with the amino- and carboxyterminals both on the cytoplasmic side. A pore domain sequence containing the hydrophobic amino acids leucine, phenylalanine, tryptophan (LFW) lies between the fifth and sixth transmembrane domains and presumably aligns with the other pore domains in the tetramer to form the potential pore. The sub-families are named for their distinguishing characteristics.¹ The TRPC (canonical) family is most homologous to the first channel in this family that was found in Drosophila. The TRPM family is named after its founding member, melastatin. The TRPV family is named after its original member, the Vanilloid receptor. TRPA family members have large numbers of Ankyrin domains. TRPP (polycystin) family members are involved in renal tubule function and TRPML (mucolipin) are unique in that they function in endosomal membranes instead of at the plasma membrane. Ankyrin repeat domains along the N-terminus of all family members facilitate tetramerization as well as interactions with other proteins. TRP cytoplasmic domains also contain binding sites for caveolin-1, phospholipase C, protein kinases G and K, calmodulin, and other signal transduction molecules on the amino- and C-terminal intracellular domains.²

Figure 1.

Topology and domain structure of TRPC6. Each of the 4 TRPC6 monomers within a TRPC6 homotetramer contains 6 transmembrane domains with the pore region (LFW, pore motif) between domains 5 and 6. Structural domains for channel assembly and protein interaction sites are located on the cytoplasmic N and C termini. Figure adapted from ref. 2 with updates.

TRP channels can be activated by a variety of signals including intracellular calcium store depletion, stretch, pressure, chemicals, temperature changes, reactive oxygen species (ROS), and a variety of surface receptors. Members of the specific subfamilies tend to be stimulated by similar stimuli, but most channels can be activated by more than one stimulus. These characteristics enable TRP channels to function within a multitude of physiologic and pathological contexts.

TRP channels and vascular permeability

An introduction to TRPC channels

TRPC channels are one of the most-well studied of the TRP family due to their ubiquitous expression in the body and particularly in endothelial cells. Therefore, we will review the TRPC family in more detail, as it provides a general model for how TRP channels function in non-excitable cells.

The family of canonical transient receptor potential channels (TRPCs) is composed of non-selective cation channels at the plasma membrane that are highly related to Drosophila TRP, the founding member of the TRP superfamily.³ Its 7 family members (TRPC1–7) can be divided into subfamilies on the basis of their amino acid sequence homology. TRPC4 and TRPC5 share <65% homology, while TRPC1 and TRPC2 (which is a non-functional pseudogene in humans⁴) are unique. TRPC3, −6, and −7 form a structural and functional subfamily, sharing 70–80% homology and are directly activated by diacylglycerol (DAG).^5-7 DAG and inositol 1,4,5-trisphosphate (IP3) are products of phosphatidylinositol 4,5-bisphosphate (PIP₂) cleavage by activated phospholipases C (PLC).⁸ Thus, it is assumed that all signaling pathways initiated by GPCR or receptor tyrosine kinase (RTK) activation that involve the activation of PLC isoforms have the potential to activate and/or modulate TRPC function.⁹

TRPC channels contain 4 N-terminal ankyrin repeats, a PLCγ binding domain, Protein Kinase G (PKG) association domains, a predicted coiled-coil region, and a putative caveolin-1 binding region, while the C-terminus displays a conserved TRP signature motif (EWKFAR), a Protein Kinase C (PKC) binding domain, and a calmodulin/IP₃ receptor binding (CIRB) domain.⁹ TRPC channels are extremely diverse due to their ability to form functional homo- and heterotetramers within 2 defined subgroups: TRPC1/4/5 and TRPC3/6/7.^10,11 TRPC1/4/5 can heterotetramerize with members of its subgroup as well as interact with Orai, a different plasma membrane Ca²⁺channel, to form calcium release activated channels (CRAC) and mediate store-operated calcium entry. In contrast, TRPC3/6/7 mediate receptor-operated calcium entry subsequent to their activation by DAG.^5,7 TRPCs can also indirectly contribute to changes in intracellular Ca²⁺ via their function as non-selective cation channels. For example, TRPCs can alter membrane potential by mediating Na⁺ influx, thereby modulating the activity of voltage-gated Ca²⁺ channels.⁹

TRP channel interactions with downstream mediators are also regulated by [Ca²⁺]. Calmodulin (CaM) and IP₃ receptor compete for binding to the CIRB domain on TRPC family members.^12-14 CaM binds only in the presence of calcium, so at low [Ca²⁺] the CIRB site is bound predominantly to the IP₃ receptor and can be competed off by CaM as the calcium concentration rises. The affinities of the CIRB site for CaM as well as the [Ca²⁺] at which binding of CaM to the CIRB is favorable vary among the different TRP channels and range over an order of magnitude.¹⁴ Therefore, the degree to which CaM or IP₃R are constitutively bound to and regulate TRP channel activity vary among the channels.¹³

Due to the multiple signaling molecules with which TRP channels directly interact, as well as the calcium ion that they permit to enter the cell, TRP channels can activate virtually every downstream signaling effector pathway that has been implicated in calcium signaling. Quite a few of these are relevant to regulating barrier function since they affect the structure and/or function of adherens and/or tight junctions. These include alterations in the actin cytoskeleton,^15-17 changes in the composition of tight junctions and adherens junctions,^18-20 alterations in the architecture of these junctions,²¹ and activation of RhoA and Rho kinase.^16,17,20,22 Generally, these phenomena are related and TRP channels activate more than one mechanism to alter permeability. In addition to acute changes in barrier function, TRP channels are also involved in regulating chronic or baseline permeability levels. For example, TRPC1 normally suppresses sphingosine kinase activity, keeping sphingosine-1-phosphate levels low, making endothelial cells more responsive to mediators such as thrombin.¹⁹ TRPC1-deficient mice have a markedly blunted response to thrombin, which can be restored by pharmacologic inhibition of sphingosine kinase.¹⁹ The signaling pathways downstream of TRP channels that are relevant to permeability are described in Fig. 2.

Figure 2.

Effector pathways from TRP channels that regulate barrier function. The ways that TRP channels influence barrier function include promoting intracellular tension by actin-myosin contraction mediated by calcium induced (1) activation of myosin light chain kinase (MLCK) through calmodulin (CaM), (2) RhoA activation of Rho kinase (ROCK) to inhibit inactivation of myosin phosphatase PP1c, (3) altering distribution of ZO-1 between tight junctions and cytoplasm, (4) endocytosis of occluding from tight junctions, and (5) direct interaction with catenins to influence their association with actin filaments.

Ca²⁺-mobilizing agonists have been shown to activate TRPC channels in endothelial cells from various vascular beds.²³ As such, TRPC channels are involved in a diverse set of vascular functions, including regulation of vascular tone and permeability, soluble mediator secretion, endothelial cell proliferation and apoptosis.^24,25

TRPC6 is expressed ubiquitously in humans, with higher expression levels in lung, placenta, ovary and spleen.²⁶ Additionally, TRPC6 expression is abundant in kidney podocytes and critical to their function. Five separate gain-of-function mutations in TRPC6 have been shown to cause familial focal and segmental glomerulosclerosis (FSGS) in the kidney due to constitutive calcium signaling²⁷ and increased current amplitudes.²⁸

Although TRPC6 is categorized as a non-selective cation channel, it is approximately 5-fold more permeable to Ca²⁺ than Na⁺.^5,29,30 TRPC6 was the first ion channel identified that is activated by DAG in a membrane-delimited fashion, independently of PKC. The DAG binding and activation site on TRPC6 is not yet known; however, it is assumed to be near one of the transmembrane domains.³¹ Other proposed mechanisms of activation besides DAG include mechanical stretch³² and exposure to reactive oxygen species.³³ Channel activity may also be fine-tuned or augmented by translocation from the cytosol to the plasma membrane,^34,35 phosphorylation,³⁶ glycosylation,²⁹ IP3 receptor binding,³⁷ and calmodulin binding.³⁸

TRPC6 regulates a multitude of physiologic processes in the body. In addition to its role in kidney function, there are numerous studies that characterize TRPC6 expression and function in the heart. These studies constitute a mounting body of evidence supporting a role for calcium influx in slowly progressive remodeling processes of the heart, such as cardiac hypertrophy induced by pressure overload and chronic hypertension.³⁹ For example, TRPC6 is upregulated in mouse hearts in response to activated calcineurin and pressure overload, as well as in failing human hearts.⁴⁰ Two conserved nuclear factor of activated T cells (NFAT) consensus sites in the promoter of the TRPC6 gene confer responsiveness to cardiac stress, thus implicating TRPC6 as a positive regulator of calcineurin-NFAT signaling and a key component of a calcium-dependent regulatory loop that drives pathologic cardiac remodeling.⁴⁰ TRPC6 has also been shown to regulate myofibroblast differentiation, as TRPC6-deficient mice display defective cardiac wound healing following injury.⁴¹

TRPC6 serves a critical function in leukocyte biology as well. TRPC6^−/− mice exhibit a blunted allergic airway response as evidenced by a decrease in blood IgE levels and a reduction in T helper cell 2 (Th2) cell secretion of IL-5 and IL-13.⁴² TRPC6 is also expressed in alveolar macrophages; TRPC6 mRNA expression is significantly increased in macrophages from chronic obstructive pulmonary disease patients.⁴³ Furthermore, this channel plays a crucial role in leukocyte chemotaxis. Neutrophils lacking TRPC6 do not migrate efficiently in response to CXCL2⁴⁴; TRPC6 was shown to be required for chemotaxis mediated by CXCR2 receptor signaling, but not that of N-formyl-methionine-leucine-phenylalanine (fMLP) receptor.⁴⁵

Finally, TRPC6 plays an extensive role in the vasculature. Vascular smooth muscle cells, which provide structural integrity for the vessel and regulate vascular tone and blood pressure, express high levels of TRPC6. TRPC6-deficient mice exhibit both increased blood pressure and enhanced smooth muscle cell contractility,⁴⁶ and patients with idiopathic pulmonary arterial hypertension display upregulation of TRPC6 in smooth muscle cells.⁴⁷ Their role in endothelial cells vis-à-vis permeability will be discussed below.

TRP channels and endothelial permeability to fluids

The blood brain barrier (BBB) is the tightest endothelial barrier to solute permeability in the body, comprised of specialized capillary endothelial cells as well as astrocyte and pericyte foot processes.⁴⁸ It is well known that shifts in endothelial cellular calcium ion concentration are required for cytoskeletal and cell adhesion molecule changes involved in promoting vascular permeability. The BBB is an obvious place to study the role of calcium channels in maintaining vascular barrier function. An immortalized mouse brain microvascular endothelial cell line, bEnd3, as well as isolated mouse brain microvessels both express TRPC 1,2,4, and 7; TRPV2 and 4; and TRPM2,3,4, and 7 by polymerase chain reaction.⁴⁹ In addition, isolated mouse brain microvessels express TRPC3, 5, and 6 as well as TRPV6.⁴⁹ Calcium flux and increased monolayer permeability were measured in response to thrombin, histamine, hypoosmolarity, and various chemicals. Response to thrombin was inhibited by pan-TRPC inhibitors, but not by the TRPV inhibitor ruthenium red.⁴⁹ Conversely, calcium flux and permeability changes in response to stimuli that activate TRPV channels (hypoosmotic stress, phorbol myristate acetate) were inhibited by ruthenium red.⁴⁹

Most of the studies on the role of TRP channels in vascular permeability have been performed in vitro and many are limited to interrogating which TRP channel is mediating increases in intracellular calcium concentration (↑[Ca²⁺]_i) in response to various stimuli. The assumption was that had this ↑[Ca²⁺]_i or changes in the cytoskeleton occurred in endothelium in vivo, there would be an increase in vascular permeability or a reduction in transendothelial electrical resistance (TEER). One such study elucidated the role of endothelial mechanosensitive TRPP2 and TRPC1 channels in mediating blood brain barrier damage in a model of traumatic brain injury by subjecting endothelial cells to cyclic stretch in vitro. They showed that siRNA silencing of either TRPP2 or TRPC1 or both led to a significant decrease in calcium flux in response to this stimulus, associated with a decrease in actin stress fiber formation.⁵⁰ However, no data on changes in actual barrier function were tested. Another study using bEnd3 monolayers measured an increase in permeability due to hypoxia and found that this was blocked by SKF-96365, a non-specific inhibitor of TRPC family channels.¹⁷ The authors proposed that TRPC channels mediated the ↑[Ca²⁺]_i required for phosphorylation of myosin light chain required for actin-myosin contraction and the increase in permeability.¹⁷

Most of what we know about TRP channels and endothelial barrier formation in the lung implicates channels of the canonical (TRPC) family in pulmonary endothelial cells. TRPC channels are by far the most studied TRP channels in endothelial cells.^8,16,51,52 Of the 3 TRPC channels that are activated directly by DAG (i.e., TRPC3/6/7), TRPC6 is the most highly expressed in several human endothelial cell types.^53,54 Endothelial TRPC6 has been demonstrated to play an important role in regulating endothelial permeability in response to various cytokines and inflammatory mediators.^16,42,55-57 Singh et al. showed that suppression of TRPC6 in human pulmonary artery endothelial cells leads to protection against increases in endothelial cell permeability in response to thrombin.⁵⁵ Similar findings were also described for bradykinin-mediated increases in endothelial permeability.⁵⁶

Maintenance of the vascular barrier is also critical in the lung, where changes in vascular permeability of clinical importance manifest as pulmonary edema, which can be life-threatening. It comes as no surprise that the role of TRP channels in maintaining barrier function has been studied most extensively in models of pulmonary edema. TRPC6 plays a key role in LPS-induced lung vascular permeability and inflammation. Investigators showed that that toll-like receptor 4 (TLR4) ligation with LPS induces DAG production and activation of TRPC6, resulting in activation of myosin light chain kinase and endothelial cell contraction, increased lung vascular permeability, and recruitment of leukocytes.¹⁶ Endothelial TRPC6, but not leukocyte TRPC6, is essential for lung ischemia reperfusion-induced edema, as irradiated TRPC6-/- mice reconstituted with wild-type bone marrow were completely protected from pulmonary vascular leakage.⁵⁷

Vascular endothelial growth factor (VEGF) also increases vascular permeability by stimulating endothelial Ca²⁺ entry. In human microvascular endothelial cells, VEGF-induced cation currents were similar to those of VEGF-mediated TRPC currents in cells expressing heterologous VEGF receptor 2 (VEGFR2) and TRPC6.⁵⁸

In addition to receptor operated TRPC6, the store operated channels TRPC1 and TRPC4 that are activated by Ca²⁺ store depletion have been implicated in pulmonary endothelial permeability.⁵⁹ Transfection of human umbilical vein endothelial cells (HUVEC) with TRPC1 cDNA enhanced the ↑[Ca²⁺]_i response as well as decreased the transendothelial electrical resistance in response to thrombin.⁶⁰ This response involves the small GTPase RhoA to promote the interaction of the inositol triphosphate receptor (IP₃R) of the endoplasmic reticulum with TRPC1 at the plasma membrane triggering ↑[Ca²⁺]_i following store depletion.²²

TRPC4^−/− mouse lungs displayed a reduction in pulmonary filtration coefficient in response to thrombin. In vitro, this was associated with a reduction in actin stress fiber formation in response to thrombin-activated PAR-1.¹⁵ Cultured endothelial cells from wild-type mice demonstrated a 50% decrease in transendothelial electrical resistance in response to thrombin; endothelial cells from TRPC4^−/− mice show only a 16% decrease.⁶¹ This implicates TRPC4 as a major contributor to the ↑[Ca²⁺]_i in response to thrombin, but raises the question of which channel(s) is responsible for the remainder.

TRPV4, a member of the vanilloid family of TRP channels, is expressed in lung vasculature, especially in the alveoli and can be activated by heat, mechanical and fluid shear, and certain lipids such as epoxyeicosatrienoic acids.⁵⁹ Chemical activation of TRPV4 in wild-type mice led to intra-alveolar edema that was not seen in TRPV4-deficient mice.⁵⁹ This edema did not seem to be associated with endothelial gaps but rather with vacuolization and membrane changes in alveolar endothelium that may be due to the actions of matrix metalloproteinases.⁶²

Pulmonary edema is clinically important because fluid in the alveoli compromises gas exchange. Yet, an important question regarding pulmonary vascular leakage is where along the vascular tree the leakage occurs. There is some evidence that vascular leakage due to activation of TRPC1 and 4 takes place in larger vessels leading to peribronchial interstitial edema that tracks secondarily to the alveolar space, while activation of TRPV4 is associated with direct permeability changes in alveolar capillaries.⁵⁹

TRP channels and epithelial permeability

Epidermis

The skin is a major barrier to water and electrolyte loss. However, epidermal barrier function is mechanistically different from simple epithelial barriers such as the gut or lining of other viscera where tight junctions form the impermeable seal along a single epithelial layer. Epidermis has an outer water-impermeable barrier: the stratum corneum. This is composed of keratin-rich dead cells and intercellular lipid. The initial response to a breach in the barrier comes from the stratum granulosum, the living layer directly below the stratum corneum. Lamellar bodies comprised of lipid are rapidly exocytosed from the epithelial cells of the stratum granulosum to form a hydrophobic barrier beneath the stratum corneum. Restoration of barrier function involves the control of this degranulation.⁶³ The second layer of the barrier is made up of the multiple tight junctions and adherens junctions of the stratified squamous epithelium. The cells of the single basal layer and the multiple layers of living cells in the stratum granulosum comprise this barrier.⁶⁴

TRPV1, V3, and V4 are expressed in keratinocytes. Denda, et al.⁶⁵ studied the role of skin surface temperature on epidermal permeability barrier in mice and men measuring transepidermal water loss following tape stripping of skin. Barrier recovery was accelerated as temperature increased from 36°C to 40°C, but then decreased above that. TRPV4 is activated by temperatures around 35°C; TRPV1 is activated at temperatures ≥ 42°C. At ambient temperature, a specific chemical activator of TRPV4 also accelerated barrier recovery, while a nonspecific inhibitor of TRPV4 retarded recovery. Conversely, capsaicin, the classic activator of TRPV1 delayed barrier recovery while a specific antagonist blocked the delay. As a control, activators of TRPV3 had no effect on barrier recovery.⁶⁵ While still somewhat indirect, these data are consistent with activation of TRPV4 improving skin barrier function below 42°C and inhibition of TRPV1 improving it above that temperature. At even lower temperatures, TRPM8 (activated below 22°C) and TRPA1 (activated below 17°C)⁶⁶ accelerate the recovery of the permeability barrier to tape stripping.^67,68 While mechanistically less than complete, these studies were all performed in vivo.

Some mechanistic insights into this phenomenon have come from in vitro studies on cultured human epidermal keratinocytes growing in monolayers. While these cultures lack the stratum corneum, they provide direct study of intercellular junctions. Activation of TRPV4 resulted in ↑[Ca²⁺]_i and promoted the formation of structural and functional intercellular junctions able to resist electric current and passage of FITC-dextran.⁶⁴ Knockdown of TRPV4 significantly impaired the development of barrier function. The adherens junction proteins E-cadherin and β-catenin were co-immunoprecipitated with TRPV4, although the nature of this interaction and their relationship in mediating junction formation was not investigated.⁶⁴ Similar results were obtained by an independent group, who also found that activation of TRPV4 upregulated expression of the tight junction proteins claudin 4 and occludin as well as increased the levels of phosphorylated atypical PKCζ, which has been associated with enhancement of tight junction barrier function.⁶⁹

Other epithelia

Despite the hundreds of studies of TRP channels in various epithelial cells, studies specifically directed at barrier function in epithelia other than skin are rare. Human fetal retinal pigment epithelial cells form tight junctions that localize to subdomains of their plasma membrane in vitro. TRPC4 and TRPM3 localize in the region of tight junctions. Barium ion, as a surrogate for Ca²⁺ that cannot be easily exported from the cell, decreased transepithelial electrical resistance in these cells in a manner that could be blocked by lanthanum ion, a nonspecific inhibitor of TRP and other channels.⁷⁰

TRPM7 has been implicated in the formation of tight junctions in the mouse urinary bladder epithelium.⁷¹ Bladders of inducible urothelium-specific TRPM7 knockout mice displayed submucosal edema and shorter length of tight junctions along the urothelium. Elevated levels of Interleukin-1β and Tumor Necrosis Factor α were also detected in the bladder wall. While the expected cation currents were significantly reduced in urothelial cells from these mice, the mechanism by which TRPM7 channels promote barrier function was not explored.⁷¹

Some of the most mechanistic studies of the roles of TRP channels in epithelial permeability have been performed in salivary gland epithelial cells. In these cells, paracellular permeability was increased via capsaicin-mediated stimulation of TRPV1, accompanied by ultrastructual changes in the tight junctions and f-actin rearrangements.²¹ TRPV1 stimulation resulted in redistribution of occludin from the junctions to the cytoplasm and myosin regulatory light chain phosphorylation through ERK1/2 activation.¹⁸ The former presumably accounted for the tight junction morphology changes, the latter for the f-actin rearrangements. More recently, these investigators extended these observations to demonstrate a role for RhoA and Rho kinase (ROCK) in the redistribution of Zonula occludens (ZO) proteins 1 and 2 in response to TRPV1 activation-induced increase in paracellular permeability.²⁰

TRP channels and leukocyte transmigration

It is clear that TRP channels of all families are involved in the inflammatory and immune responses. They are present in leukocytes, endothelial cells, and parenchymal cells and are involved in relaying signals from the environment or from cell surface receptors into the cell to promote many aspects of the inflammatory response. These aspects of TRP channel biology have been extensively reviewed.^72-76

In keeping with the theme of regulating barriers, here we will focus on the very small literature on the role of TRP channels in the migration of leukocytes across endothelial barriers. Neutrophils lacking TRPC6 do not migrate efficiently in response to CXCL2;⁴⁴ and TRPC6 was shown to be required for chemotaxis mediated by CXCR2 receptor signaling, but not that of N-formyl-methionine-leucine-phenylalanine (fMLF) receptor.⁴⁵ On the other hand, TRPC1 has been implicated in chemotaxis of neutrophils in response to fMLF.⁷⁷ To the extent that leukocyte chemotaxis can be extrapolated to transendothelial migration or plays a role in it, these TRP channels may play a role in facilitating leukocytes across endothelial barriers.

While it possible that many TRP channels play direct roles in leukocyte extravasation across endothelium and epithelium, the only channel that has been studied in this context appears to be TRPC6. As mentioned above, TRPC6 in lung endothelium was shown to be required for the increase in vascular permeability and pulmonary edema in response to intratracheal instillation of bacterial endotoxin (LPS).¹⁶ TRPC6-deficient mice and TRPC6-deficient mice reconstituted with wild-type bone marrow displayed essentially background levels of leakage when exposed to LPS. The response was returned to wild-type levels when TRPC6 expression was restored in these mice by transfection via intravenously injected liposomes.¹⁶ TRPC6 knockout mice also had marked (> 80% reduction) in leukocyte recruitment to and/or sequestration in the pulmonary capillary beds compared with control. In addition, these mice were significantly resistant to death from sepsis induced by either intraperitoneal LPS or cecal ligation and puncture.¹⁶ Sepsis is clearly an inflammatory phenomenon; however, the role of TRPC6 specifically in mediating the extravasation of leukocytes was not examined.

A direct role for TRPC6 in leukocyte extravasation was demonstrated by Weber et al.⁷⁸ For 2 and a half decades it had been known that a transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca²⁺]_i) was required for leukocyte transmigration.⁷⁹ Pharmacological chelation of endothelial Ca²⁺ during TEM results in a phenotype in which leukocytes adhere normally to the apical surface of endothelial cells, but are unable to transmigrate across.^79-83 Interestingly, the block in TEM observed upon endothelial Ca²⁺ chelation is phenotypically similar to blocking PECAM homophilic interactions with primary antibody,⁸⁴ suggesting that these 2 processes might be related.

We found that TRPC6 in endothelial cells was responsible for the ↑[Ca²⁺]_i in the endothelium required for leukocyte transendothelial migration (TEM).⁷⁸ Homophilic interactions between PECAM on the leukocyte and PECAM on the endothelial cell initiate TEM.⁸⁴ Ligation of endothelial cell PECAM recruits membrane from the lateral border recycling compartment (LBRC) to the site of TEM to provide unligated adhesion molecules and membrane surface area to facilitate TEM.^85-87 Movement of membrane from the LBRC to the site of TEM is required for efficient TEM.^85-87

TRPC6 co-localized with PECAM at the endothelial cell border and could be recruited along with PECAM to the site of TEM or to microbeads coated with anti-PECAM antibody.⁷⁸ A blockade of neutrophil TEM using anti-PECAM monoclonal antibody could be overcome with the selective TRPC6 agonist Hyp9⁸⁸ in an endothelial calcium-dependent manner. This rescue in TEM could not be replicated by the store-operated calcium influx in response to histamine. There was no effect on transendothelial electrical resistance (TEER) of the endothelial monolayer, demonstrating that increases in TEM were independent of changes in barrier function. Mechanistically, activation of TRPC6 overcame the block by anti-PECAM antibody and restored targeted recycling of the LBRC to the site of TEM. Most of the experiments of this study were performed with neutrophils and human umbilical vein endothelial cells (HUVEC). However, similar findings were obtained with monocytes.⁷⁸

Furthermore, knockdown of endogenous TRPC6 significantly impaired targeted recycling of the LBRC and TEM. This could be rescued by re-expression of TRPC6. Additionally, expression of dominant negative TRPC6 also blocked targeted recycling and TEM.⁷⁸ Leukocytes that were unable to transmigrate after interfering with TRPC6 function resembled those blocked by anti-PECAM and by other methods of disrupting LBRC function: They remained poised along the endothelial borders tightly adherent on the apical surface of endothelial cells, but unable to transmigrate.^78,84-86

This same phenomenon was seen in vivo in a murine model of dermatitis. TRPC6 knockout mice and TRPC6 knockout mice reconstituted with wild-type bone marrow were as efficient as wild-type mice at mobilizing neutrophils to the inflamed skin. However, they displayed a substantial defect in TEM. In wild-type mice or wild-type mice reconstituted with wild-type bone marrow, about 80% of the neutrophils recruited to postcapillary venules in the tissue transmigrated. In contrast, only about 25% of the neutrophils transmigrated in the mice whose endothelial cells lacked TRPC6. Moreover, similar to our findings in vitro, almost all of the neutrophils that did not transmigrate across TRPC6-deficient endothelium were seen by 3D reconstruction of confocal micrographs to be adherent to the luminal surface of the endothelium.⁷⁸

The mechanistic link between endothelial PECAM ligation and TRPC6 activation is currently under investigation. In the meantime, the availability of a specific small molecule inhibitor of TRPC6⁸⁹ raises the possibility of targeting inflammation by selectively antagonizing TRPC6 and its role in TEM.

What next?

Clearly there is much more to learn about the role of TRP channels and the maintenance of vascular and epithelial barriers. Given the ubiquitous expression of these channels, the myriad of pathways that involve calcium signaling, and the multiple receptors with which these channels interact, it would be surprising if they did not play direct and significant roles in the development and maintenance of these barriers, as well the regulation of physiologic and pathologic changes in them. Most of the vast existing literature on this large family of channels focuses on tissue expression, regulation, and electrophysiological interrogation. TRP channels are known to be involved in a variety of physiologic functions. As new genetic and pharmacologic techniques allow more selective manipulation of TRP channels, we predict that those studying barrier function will begin to examine whether and how these channels regulate specific barriers under specific (patho)physiologic conditions. No doubt, a few years from now, we will have significantly more primary research and a great deal more information on the subject.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by grants from the National Institutes of Health (R01 HL046489 and R37 HL064774 to WAM) and Predoctoral fellowships from the American Heart Association (12PRE9330014 and 14PRE18550021 to EWW).