Search Results (126)

One major challenge in cellular neuroscience is to elucidate how the accurate alignment of presynaptic release sites with postsynaptic densely clustered ligand-gated ion channels at chemical synapses is achieved upon synapse assembly. The clustering of neurotransmitter receptors at postsynaptic sites is a key moment of synaptogenesis and determinant for effective synaptic transmission. The number of the ionotropic neurotransmitter receptors at these postsynaptic sites of both excitatory and inhibitory synapses is variable and is regulated by different mechanisms, thus allowing the modulation of synaptic strength, which is essential to tune neuronal network activity. Several well-regulated processes seem to be involved, including lateral diffusion within the plasma membrane and local anchoring as well as receptor endocytosis and recycling. The molecular mechanisms implicated are numerous and were reviewed recently in great detail. The role of pre-synaptically released neurotransmitters within the complex regulatory apparatus organizing the postsynaptic site underneath presynaptic terminals is not completely understood, even less for inhibitory synapses. In this mini review article, we focus on this aspect of synapse formation, summarizing and contrasting findings on the functional role of the neurotransmitters glycine and γ-aminobutyric acid (GABA) for initiation of postsynaptic receptor clustering and regulation of Cl⁻ channel receptor numbers at inhibitory synapses gathered over the last two decades. Full article

Three-finger toxins (3FTxs) are the largest group of nonenzymatic toxins found in snake venoms. Among them, neurotoxins that target nicotinic acetylcholine receptors are the most well-studied ligands. In addition to the classical neurotoxins, several other new classes have been characterized for their structure, receptor subtype, and species selectivity. Here, we systematically analyzed over 700 amino acid sequences of three-finger neurotoxins that interact with nicotinic acetylcholine receptors. Based on the amino acid residue substitutions in the functional sites and structural features of various classes of neurotoxins, we have classified them into over 150 distinct subgroups. Currently, only a small number of typical examples representing these subgroups have been studied for their structure, function, and subtype selectivity. The functional site residues responsible for their interaction with specific receptor subtypes of several toxins are yet to be identified. The molecular details of each subgroup representative toxin with its target receptor will contribute towards the understanding of subtype- and/or interface-selectivity. Thus, this review will provide new impetus in the toxin research and pave the way for the design of potent, selective ligands for nicotinic acetylcholine receptors. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article

Olfaction has been extensively studied in the yellow fever mosquito, Aedes aegypti. This species uses its sense of smell to find blood hosts and other resources, contributing to its impact as a vector for human pathogens. Two major families of protein-coding genes, the odorant receptors (Ors) and the ionotropic receptors (Irs), provide the mosquito with sensitivities to distinct classes of volatile compounds in the antennae. Individual tuning receptors in both families require co-receptors for functionality: Orco for all Ors, and Ir8a for many Irs, especially ones that are involved in carboxylic acid detection. In Drosophila melanogaster, disruptions of Orco or Ir8a impair receptor function, tuning receptor expression, and membrane localization, leading to general anosmia. We reasoned that Orco and Ir8a might also be important for coordinated chemosensory receptor expression in the antennal sensory neurons of Ae. aegypti. To test this, we performed RNAseq and differential expression analysis in wildtype versus Orco^−/− and Ir8a^−/− mutant adult female antennae. Our analyses revealed Or and Ir tuning receptors are broadly under-expressed in Orco^−/− mutants, while a subset of tuning Irs are under-expressed in Ir8a mutants. Other chemosensory and non-chemosensory genes are also dysregulated in these mutants. Furthermore, we identify differentially expressed transcription factors including homologs of the Drosophila melanogaster Mip120 gene. These data suggest a previously unknown pleiotropic role for the Orco and Ir8a co-receptors in the coordination of expression of chemosensory receptors within the antennae of Ae. aegypti by participating in a feedback loop involving amos and members of the MMB/dREAM complex. Full article

Breast cancer is a major global health burden with the highest incidence in women, and triple-negative breast cancer (TNBC) stands out as the most malignant subtype. Effective therapeutic targets are urgently needed to develop new therapies for TNBC. Nicotinic acetylcholine receptor is a ligand-gated ion channel receptor that is associated with the advancement of multiple cancers. Notably, α9 nicotinic acetylcholine receptor (α9 nAChR) is less investigated towards its role in cancer. This study sought to clarify the significance of α9 nAChR in TNBC. Firstly, our results uncovered that the expression of CHRNA9 was notably elevated in TNBC tissues and was associated with poor prognosis of TNBC patients. Further, our data indicated that overexpression of α9 nAChR facilitated the growth of TNBC cells, via mechanisms of simultaneously activating AKT-, ERK- and STAT3-mediated proliferation and negatively regulating ferroptosis through promoting SLC7A11/GSH/GPX4 and Keap1/Nrf2/HO1 signaling. Conversely, CHRNA9 knockdown would completely reverse all this signaling, ultimately inhibiting TNBC tumor growth both in vitro and in vivo. Finally, we reported a specific polypeptide antagonist of α9 nAChR, GeXIVA[1,2] and exerted good anti-tumor effects in tumor-bearing mice of TNBC, which indicated a great potential of GeXIVA[1,2] to be further studied as a novel targeted therapy for TNBC. This study provides a scientific basis for establishing α9 nAChR as a novel therapeutic target for TNBC, which is worthy of further development in the future. Full article

The presence and function of the opioidergic system in sensory dorsal root ganglia (DRG) was demonstrated in various animal models of pain. To endorse recent functional and transcriptional evidence of opioid receptors in human DRG, this study compared morphological and transcriptional evidence in human and rat DRG using immunofluorescence confocal microscopy and mRNA transcript analysis. Specifically, it examined the neuronal expression of mu (MOR), delta (DOR), and kappa (KOR) opioid receptors, opioid peptide precursors (POMC, PENK, and PDYN), and key pain-signaling molecules. The results demonstrate abundant immunoreactivity in human DRG for key pain transduction receptors, including the thermosensitive ion channels TRPV1, TRPV4 and TRPA1, mechanosensitive PIEZO1 and PIEZO2, and the nociceptive-specific Nav1.8. They colocalized with calcitonin gene-related peptide (CGRP), a marker for peptidergic sensory neurons. Within this same subpopulation, we identified MOR, DOR, and KOR, while their ligand precursors were less abundant. Notably, the mRNA transcripts of MOR and PENK in human DRG were highest among the opioid-related genes; however, they were considerably lower than those of key pain-signaling molecules. These findings were corroborated by functional evidence in demonstrating the fentanyl-induced inhibition of voltage-gated calcium currents in rat DRG, which was antagonized by naloxone. The immunohistochemical and transcriptional demonstration of opioid receptors and their endogenous ligands in both human and rat DRG support recent electrophysiologic and in situ hybridization evidence in human DRG and confirms their potential as analgesic targets. This peripherally targeted approach has the advantage of mitigating central opioid-related side effects, endorsing the potential of future translational pain research from rodent models to humans. Full article

Background/Objectives: Despite the availability of antiepileptic drugs (AEDs) that can manage seizures, they often come with cognitive side effects. Furthermore, the role of oxidative stress and neuroinflammatory responses in epilepsy and the limitations of current AEDs necessitate exploring alternative therapeutic options. Medicinal plants, e.g., Lavandula dentata L., are rich in phenolic compounds and may provide neuroprotective and anti-inflammatory benefits. However, limited research evaluates their effectiveness in modulating neuroinflammation and histopathological changes in epilepsy models. Therefore, the current study hypothesized that treating Lavandula dentata L. extract or essential oils may reduce neuroinflammatory responses and mitigate histopathological changes in the brain, providing a natural alternative or adjunct therapy for epilepsy management. Methods: Five groups of male Wistar rats were used: control, pilocarpine-treated epileptic, valproic acid (VPA-treated epileptic), L. dentata extract, and essential oils. Numerous electrolyte levels, monoamine levels, neurotransmitter levels, and the mRNA expression of specific gate channel subtypes were evaluated in homogenate brain tissue. Additionally, histological changes in various brain regions were investigated. Results: The investigation revealed that the extract and essential oils obtained from L. dentata L. exhibited the ability to improve the modulation of electrolytes and ions across voltage- and ligand-gated ion channels. Furthermore, it was revealed that they could decrease neuronal excitability by facilitating repolarization. Moreover, L. dentata’s oil and ethanol extract re-balances T-reg/Th-17 cytokines, restoring the pro/anti-inflammatory cytokines and Treg markers, e.g., FOXP3 and CTLA-4, to their normal level. Conclusions: The present work confirms that the extract and essential oils of L. dentata L. have different activities to ameliorate the progression of histopathological alterations. Therefore, when used in conjunction with other AEDs, the extract and essential oils of L. dentata can slow the progression of epileptogenesis. Full article

Primary aldosteronism is characterised by the excessive production of aldosterone, which is a key regulator of salt metabolism, and is the most common cause of secondary hypertension. Studies have investigated the association between primary aldosteronism and genetic alterations, with pathogenic mutations being identified. This includes a glycine-to-arginine substitution at position 151 (G151R) of the G protein-activated inward rectifier potassium (K⁺) channel 4 (GIRK4), which is encoded by the KCNJ5 gene. Mutations in GIRK4 have been found to reduce the selectivity for K⁺ ions, resulting in membrane depolarisation, the activation of voltage-gated Ca²⁺ channels, and an increase in aldosterone secretion. As a result, there is an interest in identifying and exploring the mechanisms of action of small molecule modulators of wildtype (WT) and mutant channels. In order to investigate the potential modulation of homotetrameric GIRK4^WT and GIRK4^G151R channels, homology models were generated. Molecular dynamics (MD) simulations were performed, followed by a cluster analysis to extract starting structures for molecular docking. The central cavity has been previously identified as a binding site for small molecules, including natural compounds. The OliveNet^TM database, which consists of over 600 compounds from Olea europaea, was subsequently screened against the central cavity. The binding affinities and interactions of the docked ligands against the GIRK4^WT and GIRK4^G151R channels were then examined. Based on the results, luteolin-7-O-rutinoside, pheophorbide a, and corosolic acid were identified as potential lead compounds. The modulatory activity of olive-derived compounds against the WT and mutated forms of the GIRK4 channel can be evaluated further in vitro. Full article

Background/Objectives: Most of the rapid inhibitory neurotransmission in the brain is mediated through activation of the γ-aminobutyric acid (GABA) type A (GABA_A) receptor, which is a ligand-gated ion channel. GABA_A receptor activation via GABA binding allows for an intracellular influx of Cl⁻ ions, thus inducing cellular hyperpolarization. Each GABA_A receptor consists of a combination of five subunits, and several subunits have been proposed as biomarkers and therapeutic targets in cancer. Here, we show the expression of genes encoding β subunits of the GABA_A receptor, namely GABRB1, GABRB2, and GABRB3, across the four different molecular subgroups of medulloblastoma (MB), which is the most common malignant pediatric brain tumor. We also show the associations of GABA_A receptor β subunits with MB patients’ overall survival (OS). Methods: The expression of genes encoding GABA_A receptor β subunits was analyzed using a previously described dataset comprising 763 MB tumor samples. Patients were classified into high- and low-gene-expression groups, and the Kaplan–Meier estimate was used to examine the relationship between gene expression levels and patient OS. Results: High GABRB1 expression was associated with better OS within each of the four molecular subgroups. The GABRB2 gene showed higher transcript levels in Group 3 MB compared to all other subgroups, and high expression was associated with better prognosis in Group 3 tumors. GABRB3 expression was significantly higher in Group 3 and Group 4 MB, and high expression of GABRB3 genes was associated with longer OS in the sonic hedgehog (SHH) subgroup. The high expression of GABRB1, GABRB2, and GABRB3 is associated with longer patient OS in a subgroup-specific manner. Conclusions: These results indicate a role for GABA_A receptors containing β subunits in influencing MB progression. Full article

Within the phylum Cnidaria, sea anemones (class Anthozoa) express a rich diversity of ion-channel peptide modulators with biomedical applications, but corollary discoveries from jellyfish (subphylum Medusozoa) are lacking. To bridge this gap, bioactivities of previously unexplored proteinaceous and small molecular weight (~15 kDa to 5 kDa) venom components were assessed in a mouse dorsal root ganglia (DRG) high-content calcium-imaging assay, known as constellation pharmacology. While the addition of crude venom led to nonspecific cell death and Fura-2 signal leakage due to pore-forming activity, purified small molecular weight fractions of venom demonstrated three main, concentration-dependent and reversible effects on defined heterogeneous cell types found in the primary cultures of mouse DRG. These three phenotypic responses are herein referred to as phenotype A, B and C: excitatory amplification (A) or inhibition (B) of KCl-induced calcium signals, and test compound-induced disturbances to baseline calcium levels (C). Most notably, certain Alatina alata venom fractions showed phenotype A effects in all DRG neurons; Physalia physalis and Chironex fleckeri fractions predominantly showed phenotype B effects in small- and medium-diameter neurons. Finally, specific Physalia physalis and Alatina alata venom components induced direct excitatory responses (phenotype C) in glial cells. These findings demonstrate a diversity of neuroactive compounds in jellyfish venom potentially targeting a constellation of ion channels and ligand-gated receptors with broad physiological implications. Full article

Type-A γ-aminobutyric acid (GABA_A) receptors are channel proteins crucial to mediating neuronal balance in the central nervous system (CNS). The structure of GABA_A receptors allows for multiple binding sites and is key to drug development. Yet the formation mechanism of the receptor’s distinctive pentameric structure is still unknown. This study aims to investigate the role of three predominant subunits of the human GABA_A receptor in the formation of protein pentamers. Through purifying and refolding the protein fragments of the GABA_A receptor α1, β2, and γ2 subunits, the particle structures were visualised with negative staining electron microscopy (EM). To aid the analysis, AlphaFold2 was used to compare the structures. Results show that α1 and β2 subunit fragments successfully formed homo-oligomers, particularly homopentameric structures, while the predominant heteropentameric GABA_A receptor was also replicated through the combination of the three subunits. However, homopentameric structures were not observed with the γ2 subunit proteins. A comparison of the AlphaFold2 predictions and the previously obtained cryo-EM structures presents new insights into the subunits’ modular structure and polymerization status. By performing experimental and computational studies, a deeper understanding of the complex structure of GABA_A receptors is provided. Hopefully, this study can pave the way to developing novel therapeutics for neuropsychiatric diseases. Full article

Irritable bowel syndrome (IBS) is a gastrointestinal (GI) disease accompanied by changes in bowel habits without any specific cause. Gintonin is a newly isolated glycoprotein from ginseng that is a lysophosphatidic acid (LPA) receptor ligand. To investigate the efficacy and mechanisms of action of gintonin in IBS, we developed a zymosan-induced IBS murine model. In addition, electrophysiological experiments were conducted to confirm the relevance of various ion channels. In mice, gintonin restored colon length and weight to normal and decreased stool scores, whilst food intake remained constant. Colon mucosal thickness and inflammation-related tumor necrosis factor-α levels were decreased by gintonin, along with a reduction in pain-related behaviors. In addition, the fecal microbiota from gintonin-treated mice had relatively more Lactobacillaceae and Lachnospiraceae and less Bacteroidaceae than microbiota from the control mice. Moreover, gintonin inhibited transient receptor potential vanilloid (TRPV) 1 and TRPV4 associated with visceral hypersensitivity and voltage-gated Na⁺ 1.5 channels associated with GI function. These results suggest that gintonin may be one of the effective components in the treatment of IBS. Full article

Acid-sensing ion channels (ASICs), which act as proton-gating sodium channels, have garnered attention as pharmacological targets. ASIC1a isoform, notably prevalent in the central nervous system, plays an important role in synaptic plasticity, anxiety, neurodegeneration, etc. In the peripheral nervous system, ASIC1a shares prominence with ASIC3, the latter well established for its involvement in pain signaling, mechanical sensitivity, and inflammatory hyperalgesia. However, the precise contributions of ASIC1a in peripheral functions necessitate thorough investigation. To dissect the specific roles of ASICs, peptide ligands capable of modulating these channels serve as indispensable tools. Employing molecular modeling, we designed the peptide targeting ASIC1a channel from the sea anemone peptide Ugr9-1, originally targeting ASIC3. This peptide (A23K) retained an inhibitory effect on ASIC3 (IC₅₀ 9.39 µM) and exhibited an additional inhibitory effect on ASIC1a (IC₅₀ 6.72 µM) in electrophysiological experiments. A crucial interaction between the Lys23 residue of the A23K peptide and the Asp355 residue in the thumb domain of the ASIC1a channel predicted by molecular modeling was confirmed by site-directed mutagenesis of the channel. However, A23K peptide revealed a significant decrease in or loss of analgesic properties when compared to the wild-type Ugr9-1. In summary, using A23K, we show that negative modulation of the ASIC1a channel in the peripheral nervous system can compromise the efficacy of an analgesic drug. These results provide a compelling illustration of the complex balance required when developing peripheral pain treatments targeting ASICs. Full article

The human louse (Pediculus humanus) is an obligatory blood feeding ectoparasite with two ecotypes: the human body louse (Pediculus humanus humanus), a competent vector of several bacterial pathogens, and the human head louse (Pediculus humanus capitis), responsible for pediculosis and affecting millions of people around the globe. GABA (γ-aminobutyric acid) receptors, members of the cys-loop ligand gated ion channel superfamily, are among the main pharmacological targets for insecticides. In insects, there are four subunits of GABA receptors: resistant-to-dieldrin (RDL), glycin-like receptor of drosophila (GRD), ligand-gated chloride channel homologue3 (LCCH3), and 8916 are well described and form distinct phylogenetic clades revealing orthologous relationships. Our previous studies in the human body louse confirmed that subunits Phh-RDL, Phh-GRD, and Phh-LCCH3 are well clustered in their corresponding clades. In the present work, we cloned and characterized a putative new GABA receptor subunit in the human body louse that we named HoCas, for Homologous to Cys-loop α like subunit. Extending our analysis to arthropods, HoCas was found to be conserved and clustered in a new (fifth) phylogenetic clade. Interestingly, the gene encoding this subunit is ancestral and has been lost in some insect orders. Compared to the other studied GABA receptor subunits, HoCas exhibited a relatively higher expression level in all development stages and in different tissues of human body louse. These findings improved our understanding of the complex nature of GABA receptors in Pediculus humanus and more generally in arthropods. Full article

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic β cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells. Full article