A Manifesto in Defense of Pain Complexity: A Critical Review of Essential Insights in Pain Neuroscience.

Chronic pain has increasingly become a significant health challenge, not just as a symptomatic manifestation but also as a pathological condition with profound socioeconomic implications. Despite the expansion of medical interventions, the prevalence of chronic pain remains remarkably persistent, prompting a turn towards non-pharmacological treatments, such as therapeutic education, exercise, and cognitive-behavioral therapy. With the advent of cognitive neuroscience, pain is often presented as a primary output derived from the brain, aligning with Engel's Biopsychosocial Model that views disease not solely from a biological perspective but also considering psychological and social factors. This paradigm shift brings forward potential misconceptions and over-simplifications. The current review delves into the intricacies of nociception and pain perception. It questions long-standing beliefs like the cerebral-centric view of pain, the forgotten role of the peripheral nervous system in pain chronification, misconceptions around central sensitization syndromes, the controversy about the existence of a dedicated pain neuromatrix, the consciousness of the pain experience, and the possible oversight of factors beyond the nervous system. In re-evaluating these aspects, the review emphasizes the critical need for understanding the complexity of pain, urging the scientific and clinical community to move beyond reductionist perspectives and consider the multifaceted nature of this phenomenon.

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.

Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

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.

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.

NMDAR Antibodies Alter Dopamine Receptors and Cause Psychotic Behavior in Mice.

OBJECTIVE: The aim was to demonstrate that antibodies from patients with anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis alter the levels of dopamine 1 receptor (D1R) and dopamine 2 receptor (D2R) and cause psychotic-like features in mice. METHODS: Cultured rat hippocampal neurons were treated with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, and the effects on clusters of D1R and D2R were quantified. In vivo studies included 71 C57BL/6J mice that were chronically infused with CSF from patients or controls through ventricular catheters connected to subcutaneous osmotic pumps. Prepulse inhibition of the acoustic startling reflex (PPI; a marker of psychotic-like behavior), memory, locomotor activity, and the density of cell-surface and synaptic D1R, D2R, and NMDAR clusters were examined at different time points using reported techniques. RESULTS: In cultured neurons, CSF from patients, but not from controls, caused a significant decrease of cell-surface D1R and an increase of D2R clusters. In mice, CSF from patients caused a significant decrease of synaptic and total cell-surface D1R clusters and an increase of D2R clusters associated with a decrease of PPI. These effects were accompanied by memory impairment and a reduction of surface NMDARs, as reported in this model. The psychotic-like features, memory impairment, and changes in levels of D1R, D2R, and NMDAR progressively improved several days after the infusion of CSF from patients stopped. INTERPRETATION: In addition to memory deficits and reduction of NMDARs, CSF antibodies from patients with anti-NMDAR encephalitis cause reversible psychotic-like features accompanied by changes (D1R decrease, D2R increase) in cell-surface dopamine receptor clusters. ANN NEUROL 2020 ANN NEUROL 2020;88:603-613.

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.

Role of zebrafish ClC-K/barttin channels in apical kidney chloride reabsorption.

KEY POINTS: We have characterized the zebrafish clc-k and barttin proteins, demonstrating that they form a protein complex mediating chloride flux in a similar manner to their mammalian counterparts. As in mammals, in zebrafish, clc-k and barttin are basically expressed in the kidney. Contrary to what is found in mammals, in zebrafish both proteins show an apical localization in the kidney. We have generated the first knockout in zebrafish of a CLC protein. Lack of clc-k in zebrafish resulted in embryonic lethality, possibly caused by a reduction in total chloride content. As a consequence, there is an up-regulation of other chloride channels and other regulatory mechanisms such as renin or the uro-guanylin receptor in the kidney. barttin is mislocalized in vivo when clc-k is not present, indicating that there is a mutual dependence of the protein expression and localization between barttin and clc-k proteins. ABSTRACT: ClC-K/barttin channels are very important for salt transport in the kidney. This function can be clearly seen since mutations in CLCNKB or BSND cause Bartter's syndrome types III and IV, respectively. Working with the freshwater teleost zebrafish, we characterized the genes homologous to the mammalian chloride channel ClC-K and its obligate subunit barttin and we obtained and studied clc-k knockout zebrafish. The zebrafish clc-k/barttin proteins are very similar to their mammalian counterparts, and both proteins are necessary to mediate chloride currents. Localization studies indicated that both proteins are exclusively expressed in the apical membranes of zebrafish kidney tubules. Knockout of clc-k resulted in embryonic lethality. These animals showed barttin mislocalization and a reduction in whole-body chloride concentration, as well as up-regulation of the expression of other chloride channels and renin, and an increase in the kidney expression of the uroguanylin receptor. Our results indicate that apical kidney chloride reabsorption through clc-k/barttin channels is crucial for chloride homeostasis in zebrafish as it is in humans. The zebrafish model could be used as a new in vivo system to study ClC-K 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.

Anionic Phospholipids Bind to and Modulate the Activity of Human TRESK Background K+ Channel.

The background K+ channel TRESK regulates sensory neuron excitability, and changes in its function/expression contribute to neuronal hyperexcitability after injury/inflammation, making it an attractive therapeutic target for pain-related disorders. Factors that change lipid bilayer composition/properties (including volatile anesthetics, chloroform, chlorpromazine, shear stress, and cell swelling/shrinkage) modify TRESK current, but despite the importance of anionic phospholipids (e.g., PIP2) in the regulation of many ion channels, it remains unknown if membrane lipids affect TRESK function. We describe that both human and rat TRESK contain potential anionic phospholipid binding sites (apbs) in the large cytoplasmic loop, but only the human channel is able to bind to multilamellar vesicles (MLVs), enriched with anionic phospholipids, suggesting an electrostatically mediated interaction. We mapped the apbs to a short stretch of 14 amino acids in the loop, located at the membrane-cytosol interface. Disruption of electrostatic lipid-TRESK interactions inhibited hTRESK currents, while subsequent application of Folch Fraction MLVs or a PIP2 analog activated hTRESK, an effect that was absent in the rat ortholog. Strikingly, channel activation by anionic phospholipids was conferred to rTRESK by replacing the equivalent rat sequence with the human apbs. Finally, in the presence of a calcineurin inhibitor, stimulation of a Gq/11-linked GPCR reduced hTRESK current, revealing a likely inhibitory effect of membrane lipid hydrolysis on hTRESK activity. This novel regulation of hTRESK by anionic phospholipids is a characteristic of the human channel that is not present in rodent orthologs. This must be considered when extrapolating results from animal models and may open the door to the development of novel channel modulators as analgesics.

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.

Leukoencephalopathy-causing CLCN2 mutations are associated with impaired Cl- channel function and trafficking.

KEY POINTS: Characterisation of most mutations found in CLCN2 in patients with CC2L leukodystrophy show that they cause a reduction in function of the chloride channel ClC-2. GlialCAM, a regulatory subunit of ClC-2 in glial cells and involved in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC), increases the activity of a ClC-2 mutant by affecting ClC-2 gating and by stabilising the mutant at the plasma membrane. The stabilisation of ClC-2 at the plasma membrane by GlialCAM depends on its localisation at cell-cell junctions. The membrane protein MLC1, which is defective in MLC, also contributes to the stabilisation of ClC-2 at the plasma membrane, providing further support for the view that GlialCAM, MLC1 and ClC-2 form a protein complex in glial cells. ABSTRACT: Mutations in CLCN2 have been recently identified in patients suffering from a type of leukoencephalopathy involving intramyelinic oedema. Here, we characterised most of these mutations that reduce the function of the chloride channel ClC-2 and impair its plasma membrane (PM) expression. Detailed biochemical and electrophysiological analyses of the Ala500Val mutation revealed that defective gating and increased cellular and PM turnover contributed to defective A500V-ClC-2 functional expression. Co-expression of the adhesion molecule GlialCAM, which forms a tertiary complex with ClC-2 and megalencephalic leukoencephalopathy with subcortical cysts 1 (MLC1), rescued the functional expression of the mutant by modifying its gating properties. GlialCAM also restored the PM levels of the channel by impeding its turnover at the PM. This rescue required ClC-2 localisation to cell-cell junctions, since a GlialCAM mutant with compromised junctional localisation failed to rescue the impaired stability of mutant ClC-2 at the PM. Wild-type, but not mutant, ClC-2 was also stabilised by MLC1 overexpression. We suggest that leukodystrophy-causing CLCN2 mutations reduce the functional expression of ClC-2, which is partly counteracted by GlialCAM/MLC1-mediated increase in the gating and stability of the channel.

Erratum: Interaction of cochlin and mechanosensitive channel TREK-1 in trabecular meshwork cells influences the regulation of intraocular pressure.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Interaction of cochlin and mechanosensitive channel TREK-1 in trabecular meshwork cells influences the regulation of intraocular pressure.

In the eye, intraocular pressure (IOP) is tightly regulated and its persistent increase leads to ocular hypertension and glaucoma. We have previously shown that trabecular meshwork (TM) cells might detect aqueous humor fluid shear stress via interaction of the extracellular matrix (ECM) protein cochlin with the cell surface bound and stretch-activated channel TREK-1. We provide evidence here that interaction between both proteins are involved in IOP regulation. Silencing of TREK-1 in mice prevents the previously demonstrated cochlin-overexpression mediated increase in IOP. Biochemical and electrophysiological experiments demonstrate that high shear stress-induced multimeric cochlin produces a qualitatively different interaction with TREK-1 compared to monomeric cochlin. Physiological concentrations of multimeric but not monomeric cochlin reduce TREK-1 current. Results presented here indicate that the interaction of TREK-1 and cochlin play an important role for maintaining IOP homeostasis. [Corrected].

Acid-sensing ion channels detect moderate acidifications to induce ocular pain.

Sensory nerve fibers innervating the ocular anterior surface detect external stimuli producing innocuous and painful sensations. Protons are among the first mediators released by damaged cells during inflammation, tissue injury, or other chronic ophthalmic conditions. We studied whether acid-sensing ion channels (ASICs) are expressed in corneal sensory neurons and their roles in the response to moderate acidifications of the ocular surface and in pathologies producing ocular surface inflammation. Moderate acidic pH (6.6) activated ASIC-like currents in corneal sensory neurons, which were blocked by ASIC1- or ASIC3-specific toxins. Acidic pH depolarizes corneal sensory neurons to fire action potentials, an effect blocked by the ASIC3 inhibitor APETx2. 2-Guanidino-4-methylquinazoline, an ASIC3 agonist, activated a population of corneal polymodal sensory nerve fibers and significantly increased the blinking and tearing rate. The nocifensive behaviors produced by application of either a moderate acidic stimulus or ophthalmic drugs formulated in acidic solution were abolished by ASIC blockers. In a model of allergic keratoconjunctivitis, nocifensive behavior was greatly reduced by ASIC3 blockade, presumably by reducing nociceptor sensitization during the inflammatory process. Our results show that, in addition to the established role of TRPV1, ASICs play a significant role in the detection of acidic insults at the ocular surface. The identification of ASICs in corneal neurons and their alterations during different diseases 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.