Orofacial pain is a multifactorial condition that severely impacts basic functions and overall quality of life in patients, underscoring the need for non-opioid therapeutic strategies. Targeting the Transient Receptor Potential Vanilloid 1 (TRPV1) pathway has emerged as a biologically plausible approach, given its central role in nociceptive transduction and peripheral sensitization. Capsaicin, a selective TRPV1 agonist, induces receptor desensitization and has shown potential in chronic pain modulation; however, its efficacy in orofacial conditions remains unclear. This systematic review and meta-analysis aimed to evaluate the efficacy and safety of capsaicin in managing orofacial pain, compared to placebo or other pharmacological interventions. Following PRISMA guidelines, a comprehensive search of five databases (PubMed, Scopus, Web of Science, CDSR, and LILACS) was conducted without language or date restrictions. Studies involving human participants with orofacial pain treated with capsaicin were included. The protocol was registered in PROSPERO (CRD420251004538). Risk of bias was assessed using the RoB2, RoB2 crossover trial and ROBINS-I checklist, and meta-analyses were performed using random-effects models. Nine studies enrolling a total of 164 participants met the eligibility criteria and encompassed temporomandibular disorders, burning mouth syndrome, oral mucositis, trigeminal neuralgia, and neuropathic facial pain. Although placebo-controlled comparisons showed a trend toward pain reduction that did not reach statistical significance (MD = -1.87; [95% CI: -3.94, 0.19]; p = 0.08; I2 = 86%), and no significant difference was found between capsaicin concentrations (MD = 0.46; [95% CI: -2.67, 3.60]; p = 0.77, I2 = 66%), pre-post analyses of uncontrolled (single-arm) studies demonstrated a significant and clinically meaningful reduction in pain following capsaicin treatment (MD = -6.39; [95% CI: -7.40, -5.38; p < 0.001, I2 = 0%). Capsaicin also showed an acceptable safety profile: although adverse events were significantly more frequent than with control (OR = 19.84; [95% CI: 4.32-91.21]; p < 0.001, I2 = 0%), these were mild, localized, and self-limited. Capsaicin may provide clinically meaningful pain relief in selected orofacial conditions, particularly burning mouth syndrome, where uncontrolled evidence was most consistent (I2 = 0%); evidence for temporomandibular disorders was more heterogeneous and did not reach significance in pooled placebo-controlled analyses.

The soluble N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP) is essential for vesicle trafficking, coordinating trans-SNARE zippering and cis-SNARE disassembly. α-SNAP also regulates autophagy, apoptosis, calcium signaling, and AMPK activity. The hyh missense mutation M105I produces a distinctive neurodevelopmental phenotype, yet its pathogenic mechanism remains unclear. Because many α-SNAP functions rely on lipid binding, we examined whether M105I alters this property. In silico modeling revealed structural rearrangements that conceal the N-terminal hydrophobic loop, and molecular dynamics simulations predicted reduced binding free energy and weakened protein–lipid interactions. These predictions were validated in vitro and in hyh mouse brains, showing diminished membrane association, particularly at the plasma membrane. Liposome flotation assays with plasma-membrane–derived lipids confirmed that M105I directly impairs lipid binding and that membrane composition influences this interaction. Thus, defective lipid engagement emerges as a central determinant of α-SNAP dysfunction and likely contributes to the pathogenesis of hyh phenotype. Combining molecular dynamics, in vitro assays, and subcellular analyses in mouse brains reveals that the α-SNAP M105I mutation conceals a hydrophobic loop and reduces its affinity for plasma membrane lipids, uncovering the basis of the hyh phenotype.

The [Formula: see text] transporter AE4 (SLC4A9) plays a role in NaCl reabsorption and pH sensing in the kidney, and Cl--dependent fluid secretion in salivary glands. Sharing functional features with other Cl-/[Formula: see text] exchangers and Na+-[Formula: see text] cotransporters, it has been proposed that AE4 mediates Cl-/cation-[Formula: see text] exchange. Our sequence alignments and molecular dynamics (MD) analysis showed that three residues, reported as critical for transport activity in other SLC4 transporters, are conserved in AE4, suggesting similarities in their ion transport mechanism. Site-directed mutagenesis and further functional experiments showed that two out of the three conserved residues (D709 and T448) are functionally relevant, but in contrast to other SLC4 transporters, where transport was almost completely abolished, AE4 mutants conserved about 50% of transport activity. In addition, alanine scanning showed that S446A and T756A decreased transport by nearly 30%. Consistent with an additive effect of mutations at positions T756 and T448, the double mutant T756A-T448I completely abolished transport in the presence of extracellular Na+ but interestingly exhibited anion transporter activity in the presence of K+ as the main extracellular cation. MD simulations revealed that the [Formula: see text] and cation coordination site is at the interface between the transmembrane segments TM3-TM10. The interaction network was importantly disrupted in the double mutant in the presence of Na+, but it is partially conserved in the presence of K+, suggesting differences in the cation coordination. In summary, we identified the putative cation coordination site of AE4 and the critical functional role of residues T756 and T448 in its transport cycle.NEW & NOTEWORTHY AE4 is a [Formula: see text] transporter that is important for electrolyte transport and pH regulation in epithelia, which has been proposed to mediate Cl-/cation-[Formula: see text] exchange. However, critical residues sustaining transport activity are not known. In this study, we show that AE4 can coordinate [Formula: see text] and Na+ or K+ at the TM3-TM10 interface and that residues T448 and T756 are crucial for cation binding and transport cycle.

The HCO3- transporter AE4 (SLC4A9) plays a role in NaCl reabsorption and pH sensing in the kidney, and Cl--dependent fluid secretion in salivary glands. Sharing functional features with other Cl-/HCO3- exchangers and Na+-HCO3- cotransporters, it has been proposed that AE4 mediates Cl-/cation-HCO3- exchange. Our sequence alignments and molecular dynamics (MD) analysis showed that three residues, reported as critical for transport activity in other SLC4 transporters, are conserved in AE4, suggesting similarities in their ion transport mechanism. Site-directed mutagenesis and further functional experiments showed that two out of the three conserved residues (D709 and T448) are functionally relevant, but in contrast to other SLC4 transporters, where transport was almost completely abolished, AE4 mutants conserved about 50% of transport activity. In addition, alanine scanning showed that S446A and T756A decreased transport by nearly 30%. Consistent with an additive effect of mutations at positions T756 and T448, the double mutant T756A-T448I completely abolished transport in the presence of extracellular Na+ but interestingly exhibited anion transporter activity in the presence of K+ as the main extracellular cation. MD simulations revealed that the HCO(3)(-)and cation coordination site is at the interface between the transmembrane segments TM3-TM10. The interaction network was importantly disrupted in the double mutant in the presence of Na+, but it is partially conserved in the presence of K+, suggesting differences in the cation coordination. In summary, we identified the putative cation coordination site of AE4 and the critical functional role of residues T756 and T448 in its transport cycle. NEW & NOTEWORTHY AE4 is a HCO3- transporter that is important for electrolyte transport and pH regulation in epithelia, which has been proposed to mediate Cl-/cation-HCO(3)(-)exchange. However, critical residues sustaining transport activity are not known. In this study, we show that AE4 can coordinate HCO3- and Na+ or K+ at the TM3-TM10 interface and that residues T448 and T756 are crucial for cation binding and transport cycle.

TRPV1, a member of the transient receptor potential (TRP) family, is a non-selective cation channel primarily known for its role in pain perception, inflammation, and thermosensation. In mammals, it responds to noxious heat (>43C) and chemical stimuli such as capsaicin and protons. It is widely expressed in sensory neurons, notably in the dorsal root and trigeminal ganglia. However, it is also found in some non-neuronal tissues, like the skin and bladder. The human canonical variant of TRPV1 renders a protein with 839 residues. Different splice variants have been described, and some display a dominant negative effect, partially or totally inhibiting the activity of the canonical counterpart. Here, we characterize a splice variant that encodes for a channel of 850 amino acids (TRPV1850). This variant is an evolutionary novelty of catarrhine (Old World monkeys and apes) primates, incorporating an exon of 33 bp long. Both imaging of membrane expression and electrophysiological recordings suggest that TRPV1850 alone does not reach the plasma membrane. However, in cells co-expressing the canonical and TRPV1850 variants, the latter would act as a dominant negative, preventing the canonical variant from reaching the plasma membrane and rendering smaller macroscopic currents in response to capsaicin. Thus, this new isoform of the TRPV1 ion channel represents a novel form of functional regulation only present in apes and Old World monkeys. ### Competing Interest Statement The authors have declared no competing interest.

Ion channels are integral membrane proteins mediating ion flow in response to changes in their environment. Among the different types of ion channels reported to date, the super-family of TRP channels stands out since its members have been linked to many pathophysiological processes. The family comprises 6 subfamilies and 28 members in mammals, which are widely distributed throughout most tissues and organs and have an important role in several aspects of cellular physiology. It has been evidenced that abnormal expression, post-translational modifications, and channel trafficking are associated with several pathologies, such as cancer, cardiovascular disease, diabetes, and brain disorders, among others. In this review, we present an updated summary of the mechanisms involved in the subcellular trafficking of TRP channels, with a special emphasis on whether different post-translational modifications and naturally occurring mutagenesis affect both expression and trafficking. Additionally, we describe how such changes have been associated with the development and progress of diverse pathologies associated with the gain or loss of functional phenotypes. The study of these processes will not only contribute to a better understanding the role of TRP channels in the different tissues but will also present novel possible therapeutic targets in diseases where their activity is dysregulated.

Mycobacterial pathogens are the causative agents of infectious disease such as tuberculosis, which is responsible for more than 1.5 million deaths annually. To better understand the physiology of mycobacteria, we focused on determining the structure and function of MycK, an ion channel encoded by the potential tuberculosis virulence gene Rv3200c. Using the non-pathogenic model organism Mycobacterium smegmatis, we found that knockout of the MycK ortholog (MycK-KO) leads to decreased cellular ATP levels compared to WT.

The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.

Slc4a genes encode various types of transporters, including Na+-HCO3− cotransporters, Cl−/HCO3− exchangers, or Na+-driven Cl−/HCO3− exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl−/HCO3− exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl−/HCO3− exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl−/HCO3− exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl− and HCO3−-dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3− ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.

BACKGROUND:The trigeminal ganglion (TG) collects afferent sensory information from various tissues. Recent large-scale RNA sequencing of neurons of the TG and dorsal root ganglion has revealed a variety of functionally distinct neuronal subpopulations, but organ-specific information is lacking. METHODS:To link transcriptomic and tissue-specific information, we labeled small-diameter neurons of 3 specific subpopulations of the TG by local application of lipophilic carbocyanine dyes to their innervation site in the dental pulp, cornea, and meninges (dura mater). We then collected mRNA-sequencing data from fluorescent neurons. Differentially expressed genes (DEGs) were analyzed and subjected to downstream gene set enrichment analysis (GSEA), and ion channel profiling was performed. RESULTS:A total of 10,903 genes were mapped to the mouse genome (>500 reads). DEG analysis revealed 18 and 81 genes with differential expression (log 2 fold change > 2, Padj < .05) in primary afferent neurons innervating the dental pulp (dental primary afferent neurons [DPAN]) compared to those innervating the meninges (meningeal primary afferent neurons [MPAN]) and the cornea (corneal primary afferent neurons [CPAN]). We found 250 and 292 genes differentially expressed in MPAN as compared to DPAN and to CPAN, and 21 and 12 in CPAN as compared to DPAN and MPAN. Scn2b had the highest log 2 fold change when comparing DPAN versus MPAN and Mmp12 was the most prominent DEG when comparing DPAN versus CPAN and, CPAN versus MPAN. GSEA revealed genes of the immune and mitochondrial oxidative phosphorylation system for the DPAN versus MPAN comparison, cilium- and ribosome-related genes for the CPAN versus DPAN comparison, and respirasome, immune cell- and ribosome-related gene sets for the CPAN versus MPAN comparison. DEG analysis for ion channels revealed no significant differences between the neurons set except for the sodium voltage-gated channel beta subunit 2, Scn2b . However, in each tissue a few ion channels turned up with robust number of reads. In DPAN, these were Cacna1b , Trpv2 , Cnga4 , Hcn1 , and Hcn3 , in CPAN Trpa1 , Trpv1 , Cacna1a , and Kcnk13 and in MPAN Trpv2 and Scn11a . CONCLUSIONS:Our study uncovers previously unknown differences in gene expression between sensory neuron subpopulations from the dental pulp, cornea, and dura mater and provides the basis for functional studies, including the investigation of ion channel function and their suitability as targets for tissue-specific analgesia.

In a recent study, Hori and colleagues demonstrated that two specific residues located in the first ankyrin repeat of TRPV1 channels modulate the threshold for temperature activation. This study highlights the importance of considering natural diversity and comparative biology when approaching biophysical questions.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a’s ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

Transient receptor potential (TRP) channels constitute a large group of membrane receptors associated with sensory pathways in vertebrates. One of the most studied is TRPV1, a polymodal receptor tuned for detecting heat and pungent compounds. Specific inhibition of the nociceptive transduction at the peripheral nerve represents a convenient approach to pain relief. While acting as a chemoreceptor, TRPV1 shows high sensitivity and selectivity for capsaicin. In contrast to the drugs available on the market that target the inflammatory system, TRPV1 antagonists act as negative modulators of nociceptive transduction. Therefore, the development of compounds modulating TRPV1 activity has expanded dramatically over time. Experimental data suggest that most agonist and antagonist drugs interact at or near capsaicin's binding site. In particular, the properties of capsaicin's head play an essential role in modulating potency and affinity. Here, we explored a cost-efficient pipeline to predict the effects of introducing chemical modifications into capsaicin's head region. An extensive set of molecules was selected by first considering the geometrical properties of capsaicin's binding site and then molecular docking. Finally, the novel ligands were ranked by combining molecular and pharmacokinetic predictions.

TRPV5 and TRPV6 are calcium-selective ion channels expressed at the apical membrane of epithelial cells. Important for systemic calcium (Ca2+) homeostasis, these channels are considered gatekeepers of this cation transcellular transport. Intracellular Ca2+ exerts a negative control over the activity of these channels by promoting inactivation. TRPV5 and TRPV6 inactivation has been divided into fast and slow phases based on their kinetics. While slow inactivation is common to both channels, fast inactivation is characteristic of TRPV6. It has been proposed that the fast phase depends on Ca2+ binding and that the slow phase depends on the binding of the Ca2+/Calmodulin complex to the internal gate of the channels. Here, by means of structural analyses, site-directed mutagenesis, electrophysiology, and molecular dynamic simulations, we identified a specific set of amino acids and interactions that determine the inactivation kinetics of mammalian TRPV5 and TRPV6 channels. We propose that the association between the intracellular helix-loop-helix (HLH) domain and the TRP domain helix (TDh) favors the faster inactivation kinetics observed in mammalian TRPV6 channels.

Transient receptor potential (TRP) proteins are a large family of cation-selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state in TRP channels are still a matter of debate. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed phylogenetic reconstruction, sequence statistics, and structural analysis over a large set of TRP channel genes. Here, we report an exceptionally conserved set of residues. This fingerprint is composed of twelve residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. Moreover, these amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. We hypothesize that differences in the connectivity between these different groups of residues harbor the apparent differences in coupling strategies used by TRP subgroups.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.