Search Results (122)

Background: Fast excitatory transmission in the central nervous system is carried out by AMPA-type glutamate receptors. Neuronal hyperexcitability and epilepsy have been associated with the dysregulation of AMPA receptor function. Modulation of the gating kinetics of AMPA receptor function has been proposed to be a desirable target for therapy, especially when the modulation is transmembrane AMPA receptor regulatory protein (TARP)-dependent and AMPA receptor subunit composition-dependent. Methods: Eight dibenzobarrelene-based heterocycles were characterized for their effects on the human embryonic kidney cells expressing homomeric GluA1 and heteromeric GluA1/2 AMPA receptors, either alone or co-expressed with the TARPγ8 auxiliary subunit, using whole-cell patch-clamp electrophysiological recordings, and the current amplitude and kinetics of desensitization and deactivation were measured after rapid glutamate application. Results: Each chemical evaluated suppressed glutamate-induced currents via AMPA receptors and augmented both desensitization and deactivation, indicating a negative allosteric modulatory effect. The co-expression of TARPγ8 diminished, but did not eradicate, the inhibition and acceleration induced by the compounds. The observations indicate that the chemicals diminish agonist-bound open states and facilitate transitions to non-conducting states while maintaining effectiveness. Conclusions: The present study describes a specific kinetic mechanism by which dibenzobarrelene derivatives impair the function of the AMPA receptor and its dependence on auxiliary proteins. The present study provides a mechanistic understanding of AMPA receptor gating modulation and establishes a pharmacological framework for future investigations in more physiologically relevant systems. Full article

Phytophthora infestans, a devastating oomycete pathogen responsible for late blight in solanaceous crops, relies on cellulose synthase (CesA) complexes for cell wall biosynthesis and virulence. Unlike plant CesAs that form homomeric trimers, oomycete CesA complexes are hypothesized to assemble as heteromeric units, yet their structural organization remains poorly defined. Here, we employed AlphaFold-Multimer and molecular docking to resolve the structural assembly of the PiCesA1–PiCesA2–PiCesA4 heterotrimer in P. infestans and identify potential ligand-binding sites for targeted inhibition. Structural modeling revealed a conserved transmembrane architecture combined with a distinctive cytosolic organization, in which N-terminal pleckstrin homology domains play a central role in heteromeric assembly. AlphaFold-Multimer consistently predicted a stable heterotrimer stabilized by cyclic interactions between pleckstrin homology domains and glycosyltransferase-A domains, forming an extensive interface network that is spatially segregated from the conserved UDP-glucose–binding catalytic core. Structure-guided docking identified potential ligands targeting pleckstrin homology–glycosyltransferase interface regions. Notably, these sites are absent or structurally divergent in plant cellulose synthases, underscoring their potential for pathogen-selective targeting. This work advances mechanistic understanding of cellulose biosynthesis in filamentous pathogens and proposes new avenues for selective disease control in agriculture. Full article

Nicotinic acetylcholine receptors (nAChRs) are membrane-bound proteins that mediate fast synaptic transmission throughout the nervous system. A functional nAChR subtype is formed by the combination of multiple subunits arranged as homomeric or heteromeric pentamers, each with a distinct pharmacological profile. Disruption of their neurotransmission contributes to various neuropathologies, emphasizing the need for detailed knowledge of receptor structure, function, subunit composition, dynamics, and potential ligand-binding sites. However, their structural complexity as integral membrane proteins has hindered expression in mammalian cell lines and proven even more challenging to crystallize, limiting insights into ligand interactions. Understanding the molecular determinants governing nAChRs function is essential for the rational design of selective therapeutics targeting neurological disorders. The emergence of a chimeric receptor approach has dramatically improved the ability to study these important proteins and opened new avenues for high-throughput screening in drug discovery efforts. This review explains how the design of chimera constructs using soluble homologs, such as AChBP, provides researchers with an immense opportunity to investigate receptor structure–function relationships and subtype-specific properties, thereby facilitating the development of more effective treatments. Full article

Acid-sensing ion channels (ASICs), activated under acidic conditions, play a critical role in ischemic brain injury, but the detailed mechanisms and signaling pathways remain unclear. Our previous studies have shown that activation of ASIC1a channels contributes to acidosis-induced neuronal injury, partially mediated by increased calcium influx. In this study, we provide evidence that activation of ASIC2a-containing channels induces zinc influx. In cultured mouse cortical neurons, ASIC currents that were insensitive to PcTx1 inhibition were potentiated by extracellular zinc. In Chinese Hamster Ovary cells transfected with different ASIC subunits, large inward currents were recorded upon a pH drop from 7.4 to 5.0 in cells expressing homomeric ASIC1a, ASIC2a, or heteromeric ASIC1a/2a channels when normal Na⁺-rich extracellular fluid (ECF) was used. However, when ECF was modified to one containing zinc as the primary cation, the same pH drop induced an inward current only in cells expressing homomeric ASIC2a or heteromeric ASIC1a/2a, but not homomeric ASIC1a. Fluorescence imaging revealed rapid zinc influx in cells expressing ASIC2a but not ASIC1a when zinc was applied with the acidic ECF. Additionally, at pH values where ASIC2a-containing channels were activated, acid-mediated neurotoxicity was exacerbated by zinc. Thus, ASIC2a-containing channels may represent a novel pathway for zinc entry and activation of these channels might contribute to zinc-mediated neurotoxicity. Full article

Variants in CLCN3 and CLCN4, encoding the neuronal endosomal Cl⁻/H⁺ antiporters ClC-3 and ClC-4, are linked to neurodevelopmental disorders with broad phenotypic variability. Over sixty CLCN4 variants have been functionally characterized, showing gain- or loss-of-function (GoF or LoF) effects. While ClC-3 can function as a homodimer, ClC-4 depends on heterodimerization with ClC-3 for efficient endosomal trafficking. CLCN4, located on the X chromosome, exhibits diverse pathogenic outcomes: complete LoF variants often cause non-syndromic presentations in hemizygous males and are asymptomatic in heterozygous females, whereas certain missense variants with partial or complete LoF produce severe syndromic phenotypes in both sexes. Here, we demonstrate dominant effects of three CLCN4 variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes and whole-cell patch-clamp recordings in mammalian cells co-expressing both proteins via a bicistronic IRES construct. Our findings provide the first evidence of dominant-negative CLCN4 effects within ClC-3/ClC-4 complexes and establish a platform for functional analysis of additional disease-associated variants. Full article

The swallowing reflex is a highly coordinated process that is essential for safe bolus transit and airway protection. Although its neurophysiological framework has been extensively studied, the molecular mechanisms underlying reflex initiation remain incompletely understood, limiting targeted therapies for oropharyngeal dysphagia. Recent evidence implicates purinergic signaling as a key mediator of swallowing initiation, particularly through ATP release from taste buds and neuroendocrine cells in the hypopharyngeal and laryngeal mucosa. Experimental studies in mice demonstrate that water, acidic, and bitter chemical stimuli induce ATP release, activating purinergic receptors (P2X2, P2X3, heteromeric P2X2/P2X3, and P2Y1) on afferent sensory fibers. This receptor activation enhances input to the brainstem swallowing central pattern generator, initiating reflexive swallowing. Genetic ablation of purinergic receptor-expressing neurons or epithelial sentinel cells, as well as pharmacological antagonism of P2X or P2X3 receptors, markedly attenuates these responses. Furthermore, exogenous ATP or selective P2X3 agonists applied to swallowing-related mucosa evoke swallowing reflexes in an animal model, underscoring translational potential. While the precise upstream receptor mechanisms for water- and acid-induced ATP release, as well as species-specific differences, remain to be clarified, targeting purinergic pathways may represent a novel physiologically grounded therapeutic strategy for restoring swallowing function in patients with oropharyngeal dysphagia. Full article

ATP citrate lyase (ACL) is a highly conserved enzyme across eukaryotes that catalyzes the generation of cytosolic acetyl-CoA from citrate—a pivotal step linking central carbon metabolism to lipid biosynthesis. In the oleaginous yeast Yarrowia lipolytica, ACL is encoded by two genes, ACL1 and ACL2, forming a heteromeric complex that mirrors the multidomain architecture of the single-chain ACL enzymes found in mammals and plants. This conservation of catalytic architecture reflects a shared catalytic strategy across kingdoms, underscoring ACL’s fundamental role in metabolic integration. In Y. lipolytica, ACL is essential for directing mitochondrial citrate toward acetyl-CoA production and subsequent lipid accumulation. Yet, in contrast to well-characterized ACLs in animals and plants, the functional mechanisms and regulation of yeast ACL remain incompletely understood. A deeper understanding of ACL in Y. lipolytica offers not only evolutionary insights but also potential avenues for engineering lipid overproduction in microbial systems. Full article

Since colonic dopamine levels are markedly reduced during inflammatory bowel disease (IBD), we investigated how dopamine affects regulatory T-cells (Treg), which critically limit gut inflammation. Previously, we showed that the stimulation of the high-affinity dopamine receptor D₃ (Drd3) impairs suppressive Treg activity and limits their recruitment into the colon upon gut inflammation. Here we study the role of the low-affinity dopamine receptor Drd2 in Treg. We find that mice harbouring Drd2-deficient T-cells developed more severe colitis induced by dextran sodium sulphate. The stimulation of Drd2 potentiated the suppressive Treg activity and increased their ability to reach the colonic tissue. A transcriptomic analysis of intestinal mucosa from IBD patients revealed an association with increased DRD3 and reduced DRD2 expression. Bioluminescence resonance energy transfer assays revealed that Drd2 and Drd3 form a heteromer. An in situ proximity ligation assay indicated that the Drd2:Drd3 heteromer was expressed on colonic Treg, and its expression was increased upon inflammation. Using peptides analogous to the transmembrane (TM) segments from Drd2 and Drd3 in bimolecular fluorescence complementation assays, we found TM peptides able to disassemble this heteromer. The heteromer disassembly dampened the suppressive Treg activity and impaired the recruitment of Treg into the colon upon inflammation. Our findings indicate that the Drd2:Drd3 heteromer constitutes a dopamine sensor that regulates suppressive Treg activity and their colonic recruitment. Full article

Parkinson’s disease (PD) is a progressive neurological disorder that affects movement, causing symptoms such as tremors, stiffness, slowness, and balance problems due to the degeneration of dopamine-producing neurons in the brain. Nowadays there is no cure for PD. Alpha synuclein (α-syn) aggregates, which are a hallmark of PD, are known to induce microglial activation, specifically the detrimental M1 microglial phenotype, which contributes to neuroinflammation and disease progression. Cannabinoid receptor 2 (CB₂R) activation has been shown to counteract neuroinflammation. CB₂R is able to interact with N-methyl-D-aspartate (NMDA) receptors (NMDAR), which has also attracted attention in PD research due to its role in excitotoxicity. Here we aimed to study the interaction between CB₂R and NMDAR in a PD context in rat tissue. We observed that α-syn fibrils alter CB₂R activation and CB₂R-NMDAR heteromerization in a heterologous expression system. Furthermore, activation of CB₂R counteracted NMDAR signaling. In microglia, α-syn fibrils decreased CB₂R-NMDAR heteromer expression while increasing CB₂R signaling. Importantly, CB₂R activation counteracted the α-syn fibrils-induced increase in M1-activated microglia, while it favored the polarization of microglia to the beneficial M2 phenotype. These results reinforce the idea of using cannabinoids for treating PD, as they provide not only the anti-inflammatory effects of cannabinoids but also counteract the detrimental increase in NMDAR signaling present in this disease. Full article

Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange but, when impaired, can result in life-threatening hypoxemia. Moreover, under conditions of generalized alveolar hypoxia, HPV can result in pulmonary hypertension. Voltage-gated K⁺ channels (K_v channels) are key to HPV: a change in the intracellular hydrogen peroxide (H₂O₂) levels during acute hypoxia is assumed to modulate these channels’ activity to trigger HPV. However, there are longstanding conflicting findings on whether H₂O₂ inhibits or activates K_v channels. Therefore, we hypothesized that H₂O₂ affects K_v channels depending on the experimental conditions, i.e., the H₂O₂ concentration, the channel’s subunit configuration or the experimental clamping potential in electrophysiological recordings. Therefore, cRNAs encoding the K_v1.5 channel and the auxiliary K_vβ subunits (K_vβ1.1, K_vβ1.4) were generated via in vitro transcription before being injected into Xenopus laevis oocytes for heterologous expression. The K⁺ currents of homomeric (Kv1.5) or heteromeric (K_v1.5/K_vβ1.1 or K_v1.5/K_vβ1.4) channels were assessed by two-electrode voltage clamp. The response of the K_v channels to H₂O₂ was markedly dependent on (a) the clamping potential, (b) the H₂O₂ concentration, and (c) the K_v channel’s subunit composition. In conclusion, our data highlight the importance of the choice of experimental conditions when assessing the H₂O₂ sensitivity of K_v channels in the context of HPV, thus providing an explanation for the long-lasting controversial findings reported in the literature. Full article

A de novo missense variant in KCNH3 has been identified in a patient with neurological symptoms including seizures. Here, we confirm the previously reported loss-of-function features for the associated Kv12.2 mutant A371V and investigate the underlying mechanism. Loss of function was not rescued by low temperature during channel biogenesis. Elevated external K⁺ reduced the rectification of Kv12.2 conductance as predicted by the GHK current equation, allowing the detection of currents mediated by homomeric A371V Kv12.2 channels and a detailed biophysical analysis of the mutant. Compared to wild-type, the voltage dependences of activation and deactivation of A371V Kv12.2 channels were shifted in the positive direction by 15 to 20 mV. Moreover, A371V Kv12.2 channels exhibited accelerated inactivation kinetics combined with a dramatic negative shift in the voltage dependence of inactivation by more than 100 mV. Even in heteromeric wild-type + A371V Kv12.2 channels, inactivation was enhanced, leading to a significant current reduction at physiological potentials. Our Kv12.2 data show similarities to Kv11 channels regarding C-type inactivation and differences regarding the sensitivity to external K⁺ and pharmacological inhibition of inactivation. The gating modification caused by the A371V amino acid substitution in Kv12.2 renders loss of function voltage-dependent, with a possible impact on neuronal excitability and firing behavior. Full article

Background/Objectives: Kv2 channels have important conducting and nonconducting functions and are regulated by their co-assembly with ‘silent’ Kv subunits, including Kv9.1. Kv9.1 is co-expressed with Kv2 channels in sensory neurons, and a common allele that changes Ile489 to Val in human Kv9.1 is associated with pain hypersensitivity in patients. The mechanism responsible for this association remains unknown, but we hypothesise that these two variants differ in their regulation of Kv2.1 properties, and this is what we set out to test. Methods: Expression was carried out using HEK293 cells, OHeLa cells, and primary cultures of hippocampal neurons, and the biophysical and trafficking properties of homomeric and heteromeric channels were assessed by confocal fluorescence microscopy and patch clamp analysis. Results: Both Kv9.1Ile and Kv9.1Val were retained within the endoplasmic reticulum when expressed individually, but when co-expressed with Kv2.1, they co-localised with Kv2.1 within the surface clusters. Both variants reduced the surface expression of Kv2.1 channels and the size of channel clusters, with Kv9.1Val producing a greater reduction in surface expression in both the HeLa cells and neurons. They both caused a similar hyperpolarising shift in the voltage dependence of channel activation and inactivation. Concatamers of Kv2.1 and Kv9.1 suggested that both 3:1 and 2:2 ratios of Kv2.1 to Kv9.1 were permitted, although 2:2 resulted in lower surface expression and function. Conclusions: The Ile489Val substitution in Kv9.1 does not disrupt its ability to co-assemble with Kv2 channels, nor its effects on the voltage-dependence of channel gating, but it did produce a greater reduction in the Kv2.1 surface expression, suggesting that this underlies its association with pain hypersensitivity. Full article

P2X receptors are unspecific cation channels activated by ATP. They are expressed in various tissues and found in neuronal and immune cells. In mammals, seven subunits are described, which can assemble into homomeric and heteromeric trimers. P2X2 receptors play important roles in cochlear adaptation to elevated sound levels. Three mutations causing inherited progressive hearing loss have been identified. These mutations localize to the transmembrane domain 1 (V60L), the transmembrane domain 2 (G353R) and a β-sheet linking the ATP binding site to the pore (D273Y). Herein, mutations were studied in human homomeric P2X2 as well as in heteromeric P2X2/3 receptors. We measured their binding of a fluorescently labeled ATP derivative (fATP) and characterized the constructs using the patch-clamp technique. The conclusions from our results are as follows: 1. The mutations V60L and G353R show robust localization on the plasma membrane and binding of fATP, whereas the mutant D273Y has no binding to fATP. 2. The mutation V60L has an increased affinity to fATP compared with the wildtype. 3. The expression of hP2X2 V60L channels reduces cell viability, which may support its role in the pathogenesis of hearing loss. 4. All mutant P2X2 subunits can assemble into P2X2/3 heteromeric channels with distinct phenotypes. Full article

Cyanide (CN) is a potent, fast-acting toxicant that impacts endogenous biomolecules in the nervous system, including acid-sensing ion channels (ASICs), which play a vital role in various neurological and psychological conditions. Here, we demonstrate that CN rapidly potentiates ASIC currents in cultured mouse cortical neurons in a dose-dependent manner while causing a leftward shift in the pH dose–response curve. Notably, this potentiation was unaffected by a 30-min CN treatment or the presence of ATP in the recording pipette. Further investigations into the role of zinc revealed that TPEN, a high-affinity zinc chelator, did not enhance ASIC currents following CN pretreatment, nor did CN influence the potentiation of ASIC currents induced by TPEN. Low-affinity zinc blocked the potentiation of ASIC currents by CN. CN potentiated ASIC currents in cortical neurons from ASIC2 but not from ASIC1a knockout mice. In experiments with CHO cells expressing homomeric ASIC1a and heteromeric ASIC1a/2, CN potentiated ASIC1a currents but had no effect on homomeric ASIC1b, ASIC2a, or ASIC3 channels. Mutating lysine 133 (K133) to arginine (R) in the extracellular domain of ASIC1a abolished CN’s effect, suggesting that CN potentiates ASIC1a currents primarily via high-affinity zinc binding, with K133 being critical for this modulation. Full article

Isoxazole carboxamide derivatives are intriguing modulators of ionotropic glutamate receptors; more specifically, their prospective analgesic activities based on non-opioid pathways have sparked widespread research. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, especially Ca²⁺-permeable subtypes that are highly expressed in the spinal dorsal horn, play a critical role in nociceptive transmission and inflammatory pain. Herein, the neuromodulatory effects of these derivatives on AMPA receptor activity have been studied, focusing on their potential as modulators of AMPA receptors, a target implicated in pain and neurological disorders. The whole-cell patch clamp technique for electrophysiological recordings was used to investigate the effect of twelve isoxazole-4-carboxamide derivatives (CIC-1-12) on AMPA receptors’ whole-cell currents and kinetics, including deactivation and desensitization. The isoxazole-4-carboxamide derivatives tested as inhibitors of AMPA receptor activity were very potent, with an 8-fold inhibition by CIC-1 and a 7.8-fold reduction by CIC-2. Additionally, these compounds profoundly altered the biophysical gating properties of both homomeric and heteromeric receptor subunits. These findings emphasize the therapeutic promise of isoxazole-4-carboxamide derivatives due to their potential as AMPA receptor modulators. Their ability to affect receptor activity and gating properties makes them promising candidates for future treatments for controlling pain. Full article