Differential Gene and Protein Expression of Conjunctival Bleb Hyperfibrosis in Early Failure of Glaucoma Surgery.

The early failure of glaucoma surgery is mainly caused by over-fibrosis at the subconjunctival space, causing obliteration of the filtration bleb. Because fibrosis has a suspected basis of genetic predisposition, we have undertaken a prospective study to identify upregulated profibrotic genes in a population of glaucoma patients with signs of conjunctival fibrosis and early postoperative surgical failure. Clinical data of re-operated fibrosis patients, hyperfibrosis patients who re-operated more than once in a short time, and control patients with no fibrosis were recorded and analyzed at each follow-up visit. Conjunctival-Tenon surgical specimens were obtained intraoperatively to evaluate the local expression of a panel of genes potentially associated with fibrosis. In order to correlate gene expression signatures with protein levels, we quantified secreted proteins in primary cultures of fibroblasts from patients. Expression of VEGFA, CXCL8, MYC, and CDKN1A was induced in the conjunctiva of hyperfibrosis patients. VEGFA and IL8 protein levels were also increased in fibroblast supernatants. We propose that an increase in these proteins could be useful in detecting conjunctival fibrosis in glaucoma patients undergoing filtering surgery. Molecular markers could be crucial for early detection of patients at high risk of failure of filtration surgery, leading to more optimal and personalized treatments.

TRESK background K+ channel deletion selectively uncovers enhanced mechanical and cold sensitivity.

KEY POINTS: TRESK background K+ channel is expressed in sensory neurons and acts as a brake to reduce neuronal activation. Deletion of the channel enhances the excitability of nociceptors. Skin nociceptive C-fibres show an enhanced activation by cold and mechanical stimulation in TRESK knockout animals. Channel deletion selectively enhances mechanical and cold sensitivity in mice, without altering sensitivity to heat. These results indicate that the channel regulates the excitability of specific neuronal subpopulations involved in mechanosensitivity and cold-sensing. ABSTRACT: Background potassium-permeable ion channels play a critical role in tuning the excitability of nociceptors, yet the precise role played by different subsets of channels is not fully understood. Decreases in TRESK (TWIK-related spinal cord K+ channel) expression/function enhance excitability of sensory neurons, but its role in somatosensory perception and nociception is poorly understood. Here, we used a TRESK knockout (KO) mouse to address these questions. We show that TRESK regulates the sensitivity of sensory neurons in a modality-specific manner, contributing to mechanical and cold sensitivity but without any effect on heat sensitivity. Nociceptive neurons isolated from TRESK KO mice show a decreased threshold for activation and skin nociceptive C-fibres show an enhanced activation by cold and mechanical stimulation that was also observed in behavioural tests in vivo. TRESK is also involved in osmotic pain and in early phases of formalin-induced inflammatory pain, but not in the development of mechanical and heat hyperalgesia during chronic pain. In contrast, mice lacking TRESK present cold allodynia that is not further enhanced by oxaliplatin. In summary, genetic removal of TRESK uncovers enhanced mechanical and cold sensitivity, indicating that the channel regulates the excitability of specific neuronal subpopulations involved in mechanosensitivity and cold-sensing, acting as a brake to prevent activation by innocuous stimuli.

The Background K+ Channel TRESK in Sensory Physiology and Pain.

TRESK belongs to the K2P family of potassium channels, also known as background or leak potassium channels due to their biophysical properties and their role regulating membrane potential of cells. Several studies to date have highlighted the role of TRESK in regulating the excitability of specific subtypes of sensory neurons. These findings suggest TRESK could be involved in pain sensitivity. Here, we review the different evidence available that involves the channel in pain and sensory perception, from studies knocking out the channel or overexpressing it to identified mutations that link the channel to migraine pain. In addition, the therapeutic possibilities are discussed, as targeting the channel seems an interesting therapeutic approach to reduce nociceptor activation and to decrease pain.

Caveolar targeting links Kv1.3 with the insulin-dependent adipocyte physiology.

The voltage-dependent potassium channel Kv1.3 participates in peripheral insulin sensitivity. Genetic ablation of Kv1.3 triggers resistance to diet-induced weight gain, thereby pointing to this protein as a pharmacological target for obesity and associated type II diabetes. However, this role is under intense debate because Kv1.3 expression in adipose tissue raises controversy. We demonstrated that Kv1.3 is expressed in white adipose tissue from humans and rodents. Moreover, other channels, such as Kv1.1, Kv1.2, Kv1.4 and especially Kv1.5, from the same Shaker family are also present. Although elevated insulin levels and adipogenesis remodel the Kv phenotype, which could lead to multiple heteromeric complexes, Kv1.3 markedly participates in the insulin-dependent regulation of glucose uptake in mature adipocytes. Adipocyte differentiation increased the expression of Kv1.3, which is targeted to caveolae by molecular interactions with caveolin 1. Using a caveolin 1-deficient 3T3-L1 adipocyte cell line, we demonstrated that the localization of Kv1.3 in caveolar raft structures is important for proper insulin signaling. Insulin-dependent phosphorylation of the channel occurs at the onset of insulin-mediated signaling. However, when Kv1.3 was spatially outside of these lipid microdomains, impaired phosphorylation was exhibited. Our data shed light on the putative role of Kv1.3 in weight gain and insulin-dependent responses contributing to knowledge about adipocyte physiology.

Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

Involvement of potassium channels in the progression of cancer to a more malignant phenotype.

Potassium channels are a diverse group of pore-forming transmembrane proteins that selectively facilitate potassium flow through an electrochemical gradient. They participate in the control of the membrane potential and cell excitability in addition to different cell functions such as cell volume regulation, proliferation, cell migration, angiogenesis as well as apoptosis. Because these physiological processes are essential for the correct cell function, K+ channels have been associated with a growing number of diseases including cancer. In fact, different K+ channel families such as the voltage-gated K+ channels, the ether à-go-go K+ channels, the two pore domain K+ channels and the Ca2+-activated K+ channels have been associated to tumor biology. Potassium channels have a role in neoplastic cell-cycle progression and their expression has been found abnormal in many types of tumors and cancer cells. In addition, the expression and activity of specific K+ channels have shown a significant correlation with the tumor malignancy grade. The aim of this overview is to summarize published data on K+ channels that exhibit oncogenic properties and have been linked to a more malignant cancer phenotype. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

A non-canonical di-acidic signal at the C-terminus of Kv1.3 determines anterograde trafficking and surface expression.

Impairment of Kv1.3 expression at the cell membrane in leukocytes and sensory neuron contributes to the pathophysiology of autoimmune diseases and sensory syndromes. Molecular mechanisms underlying Kv1.3 channel trafficking to the plasma membrane remain elusive. We report a novel non-canonical di-acidic signal (E483/484) at the C-terminus of Kv1.3 essential for anterograde transport and surface expression. Notably, homologous motifs are conserved in neuronal Kv1 and Shaker channels. Biochemical analysis revealed interactions with the Sec24 subunit of the coat protein complex II. Disruption of this complex retains the channel at the endoplasmic reticulum. A molecular model of the Kv1.3-Sec24a complex suggests salt-bridges between the di-acidic E483/484 motif in Kv1.3 and the di-basic R750/752 sequence in Sec24. These findings identify a previously unrecognized motif of Kv channels essential for their expression on the cell surface. Our results contribute to our understanding of how Kv1 channels target to the cell membrane, and provide new therapeutic strategies for the treatment of pathological conditions.

Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology.

OBJECTIVE: Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. APPROACH AND RESULTS: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. CONCLUSIONS: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.

Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma.

The trabecular meshwork tissue controls the drainage of the aqueous humor of the eye. A dysfunctional trabecular meshwork leads to an altered fluid resistance, which results in increased intraocular pressure (IOP). IOP is the major risk factor of glaucoma, the second-leading cause of blindness in the developed world. In the search for genes altered by glaucomatous insults, we identified angiopoietin-like7 (ANGPTL7), a member of the ANGPTL family. Although structurally related to the angiopoietins, ANGPTL7's function is poorly understood. Because ANGPTL7 is secreted and because extracellular matrix (ECM) deposition and organization is critical for aqueous humor resistance, we investigated the effect of ANGPTL7 on relevant trabecular meshwork ECM genes and proteins. We find that overexpression of ANGPTL7 in primary human trabecular meshwork cells altered the expression of fibronectin, collagens type I, IV & V, myocilin, versican, and MMP1. ANGPTL7 also interfered with the fibrillar assembly of fibronectin. Finally, we find that silencing ANGPTL7 during the glucocorticoid insult significantly affected the expression of other steroid-responsive proteins. These results indicate that ANGPTL7 modulates the trabecular meshwork's ECM as well as the response of this tissue to steroids. Together with previous findings, these properties strengthen ANGPTL7's candidacy for the regulation of IOP and glaucoma.

Targeting of Kv7.5 (KCNQ5)/KCNE channels to surface microdomains of cell membranes.

BACKGROUND: Kv7.5 (KCNQ5) channels conduct M-type potassium currents in the brain, are expressed in skeletal muscle, and contribute to vascular muscle tone. METHODS: We coexpressed Kv7.5 and KCNE1-3 peptides in HEK293 cells and then analyzed their association using electrophysiology and co-immunoprecipitation, assessed localization using confocal microscopy, examined targeting of the oligomeric channels to cholesterol-rich membrane surface microdomains using lipid raft isolation, and evaluated their membrane dynamics using fluorescence recovery after photobleaching (FRAP). RESULTS: Kv7.5 forms oligomeric channels specifically with KCNE1 and KCNE3. The expression of Kv7.5 targeted to cholesterol-rich membrane surface microdomains was very low. Oligomeric Kv7.5/KCNE1 and Kv7.5/KCNE3 channels did not localize to lipid rafts. However, Kv7.5 association impaired KCNE3 expression in lipid raft microdomains. CONCLUSIONS: Our results indicate that Kv7.5 contributes to the spatial regulation of KCNE3. This new scenario could greatly assist in determining the physiological relevance of putative KCNE3 interactions in nerve and muscle.

Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel.

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl- currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

Proton Sensing on the Ocular Surface: Implications in Eye Pain.

Protons reaching the eyeball from exogenous acidic substances or released from damaged cells during inflammation, immune cells, after tissue injury or during chronic ophthalmic conditions, activate or modulate ion channels present in sensory nerve fibers that innervate the ocular anterior surface. Their identification as well as their role during disease is critical for the understanding of sensory ocular pathophysiology. They are likely to mediate some of the discomfort sensations accompanying several ophthalmic formulations and may represent novel targets for the development of new therapeutics for ocular pathologies. Among the ion channels expressed in trigeminal nociceptors innervating the anterior surface of the eye (cornea and conjunctiva) and annex ocular structures (eyelids), members of the TRP and ASIC families play a critical role in ocular acidic pain. Low pH (pH 6) activates TRPV1, a polymodal ion channel also activated by heat, capsaicin and hyperosmolar conditions. ASIC1, ASIC3 and heteromeric ASIC1/ASIC3 channels present in ocular nerve terminals are activated at pH 7.2-6.5, inducing pain by moderate acidifications of the ocular surface. These channels, together with TRPA1, are involved in acute ocular pain, as well as in painful sensations during allergic keratoconjunctivitis or other ophthalmic conditions, as blocking or reducing channel expression ameliorates ocular pain. TRPV1, TRPA1 and other ion channels are also present in corneal and conjunctival cells, promoting inflammation of the ocular surface after injury. In addition to the above-mentioned ion channels, members of the K2P and P2X ion channel families are also expressed in trigeminal neurons, however, their role in ocular pain remains unclear to date. In this report, these and other ion channels and receptors involved in acid sensing during ocular pathologies and pain are reviewed.

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting.

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co-express KCNQ1 with KCNE1-5 proteins. KCNQ1 may co-associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K(+) channels in sphingolipid-cholesterol-enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK-293 cells were transiently transfected with KCNQ1 and KCNE1-5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co-localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3-5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation.

The unconventional biogenesis of Kv7.1-KCNE1 complexes.

The potassium channel Kv7.1 associates with the KCNE1 regulatory subunit to trigger cardiac I Ks currents. Although the Kv7.1/KCNE1 complex has received much attention, the subcellular compartment hosting the assembly is the subject of ongoing debate. Evidence suggests that the complex forms either earlier in the endoplasmic reticulum or directly at the plasma membrane. Kv7.1 and KCNE1 mutations, responsible for long QT syndromes, impair association and traffic, thereby altering I Ks currents. We found that Kv7.1 and KCNE1 do not assemble in the first stages of their biogenesis. Data support an unconventional secretory pathway for Kv7.1-KCNE1 that bypasses Golgi. This route targets channels to endoplasmic reticulum-plasma membrane junctions, where Kv7.1-KCNE1 assemble. This mechanism helps to resolve the ongoing controversy about the subcellular compartment hosting the association. Our results also provide new insights into I Ks channel localization at endoplasmic reticulum-plasma membrane junctions, highlighting an alternative anterograde trafficking mechanism for oligomeric ion channels.

Author Correction: The LRRC8-mediated volume-regulated anion channel is altered in glaucoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Individual molecular response to elevated intraocular pressure in perfused postmortem human eyes.

Elevated intraocular pressure (IOP) is the major risk factor for glaucoma. In the clinic, the response to elevated pressure and thus the risk for development of glaucoma differs among individuals. We took advantage of our ability to subject postmortem human eyes from the same individual to physiological and elevated pressure in a perfused outflow model and compared individual patterns of gene expression under pressure. The architecture of the trabecular meshwork, tissue responsible for the maintenance of IOP, was conserved. We performed two sets of experiments. The first set (n = 5, 10 eyes) used Affymetrix GeneChips, identified the 20 most pressure-altered genes in each individual, and compared their pressure response in the other four. The second set (n = 5, 10 eyes) selected 21 relevant trabecular meshwork genes and examined, by real-time TaqMan-PCR, the rank of their abundance and of their pressure differential expression in each individual. The majority of the up- and downregulated top-changers of each individual showed an individual response trend. Few genes were general responders. Individual responders included STATH, FBN2, TF, OGN, IL6, IGF1, CRYAB, and ELAM1 (marker for glaucoma). General responders included MMP1, MMP10, CXCL2, and PDPN. In addition, we found that although the relative abundance of selected genes was very similar among nonstressed individuals, the response to pressure of those same genes had a marked individual component. Our results offer the first molecular insight on the variation of the individual response to IOP observed in the clinical setting.

Functional implications of KCNE subunit expression for the Kv7.5 (KCNQ5) channel.

Kv7 (KCNQ) proteins form a family of voltage-gated potassium channels that is comprised of five members, Kv7.1-Kv7.5. While Kv7.1 is crucial in the heart, the Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels contribute to the M-current in the nervous system. In addition to the brain, Kv7.5 is expressed in skeletal and smooth muscle, where its physiological role is currently under evaluation. Kv7 associations with KCNE accessory subunits (KCNE1-5) enhance channel diversity and their interaction provides mechanisms to respond to a variety of stimuli. KCNE peptides control the surface expression, voltage-dependence, kinetics of gating, unitary conductance, ion selectivity and pharmacology of several channels. KCNE subunits have been primarily studied in the heart; however, their activity in the brain and in many other tissues is being increasingly recognized. Here, we found that Kv7.5 and KCNE subunits are present in myoblasts. Therefore, oligomeric associations may underlie some Kv7.5 functional diversity in skeletal muscle. An extensive study in Xenopus oocytes and HEK-293 cells demonstrates that KCNE1 and KCNE3, but none of the other KCNE subunits, affect Kv7.5 currents. While KCNE1 slows activation and suppresses inward rectification, KCNE3 drastically inhibits Kv7.5 currents. In addition, KCNE1 increases Kv7.5 currents in HEK cells. Changes in gating and amplitude indicate functional interactions. Our results have physiological relevance since Kv7.5 is abundant in skeletal and smooth muscle and its association with KCNE peptides may fine-tune cellular responses.

Evidence for a calcification process in the trabecular meshwork.

The human trabecular meshwork (TM) expresses many genes that have been associated with physiological (bone, cartilage, teeth) and pathological (vascular systems, kidney) calcification. In particular, the TM highly expresses the inhibitor of calcification Matrix Gla (MGP) gene, which encodes a vitamin K-dependent protein that requires post-translational activation to inhibit the formation of calcium precipitates. TM cells have high activity of the activating gamma-carboxylase enzyme and produce active MGP. Silencing MGP increases the activity of alkaline phosphatase (ALP), an enzyme of the matrix vesicles and marker of calcification. Overexpressing MGP reduces the ALP activity induced by bone morphogenetic 2 (BMP2), a potent inducer of calcification. In this review we gathered evidence for the existence of a mineralization process in the TM. We selected twenty regulatory calcification genes, reviewed their functions in their original tissues and looked at their relative abundance in the TM by heat maps derived from existing microarrays. Although results are not yet fully conclusive and more experiments are needed, examining TM expression in the light of the calcification literature brings up many similarities. One such parallel is the role of mechanical forces in bone induction and the high levels of mineralization inhibitors found in the constantly mechanically stressed TM. During the next few years, examination of other calcification-related regulatory genes and pathways, as well as morphological examination of knockout animals, would help to elucidate the relevance of a calcification process to TM's overall function.

The LRRC8-mediated volume-regulated anion channel is altered in glaucoma.

Regulation of cellular volume is an essential process to balance volume changes during cell proliferation and migration or when intracellular osmolality increases due to transepithelial transport. We previously characterized the key role of volume-regulated anion channels (VRAC) in the modulation of the volume of trabecular meshwork (TM) cells and, in turn, the aqueous humour (AH) outflow from the eye. The balance between the secretion and the drainage of AH determines the intraocular pressure (IOP) that is the major casual risk factor for glaucoma. Glaucoma is an ocular disease that causes irreversible blindness due to the degeneration of retinal ganglion cells. The recent identification of Leucine-Rich Repeat-Containing 8 (LRRC8A-E) proteins as the molecular components of VRAC opens the field to elucidate their function in the physiology of TM and glaucoma. Human TM cells derived from non-glaucomatous donors and from open-angle glaucoma patients were used to determine the expression and the functional activity of LRRC8-mediated channels. Expression levels of LRRC8A-E subunits were decreased in HTM glaucomatous cells compared to normotensive HTM cells. Consequently, the activity of VRAC currents and volume regulation of TM cells were significantly affected. Impaired cell volume regulation will likely contribute to altered aqueous outflow and intraocular pressure.

Pyrethroids inhibit K2P channels and activate sensory neurons: basis of insecticide-induced paraesthesias.

Pyrethroid insecticides are widely used for pest control in agriculture or in human public health commonly as a topical treatment for scabies and head lice. Exposure to pyrethroids such as permethrin or tetramethrin (TM) causes sensory alterations such as transient pain, burning, stinging sensations, and paraesthesias. Despite the well-known effects of pyrethroids on sodium channels, actions on other channels that control sensory neuron excitability are less studied. Given the role of 2-pore domain potassium (K2P) channels in modulating sensory neuron excitability and firing, both in physiological and pathological conditions, we examined the effect of pyrethroids on K2P channels mainly expressed in sensory neurons. Through electrophysiological and calcium imaging experiments, we show that a high percentage of TM-responding neurons were nociceptors, which were also activated by TRPA1 and/or TRPV1 agonists. This pyrethroid also activated and enhanced the excitability of peripheral saphenous nerve fibers. Pyrethroids produced a significant inhibition of native TRESK, TRAAK, TREK-1, and TREK-2 currents. Similar effects were found in transfected HEK293 cells. At the behavioral level, intradermal TM injection in the mouse paw produced nocifensive responses and caused mechanical allodynia, demonstrating that the effects seen on nociceptors in culture lead to pain-associated behaviors in vivo. In TRESK knockout mice, pain-associated behaviors elicited by TM were enhanced, providing further evidence for a role of this channel in preventing excessive neuronal activation. Our results indicate that inhibition of K2P channels facilitates sensory neuron activation and increases their excitability. These effects contribute to the generation of paraesthesias and pain after pyrethroid exposure.