Endocannabinoid regulation of inward rectifier potassium (Kir) channels.

The inward rectifier potassium channel Kir2.1 (KCNJ2) is an important regulator of resting membrane potential in both excitable and non-excitable cells. The functions of Kir2.1 channels are dependent on their lipid environment, including the availability of PI(4,5)P2, secondary anionic lipids, cholesterol and long-chain fatty acids acyl coenzyme A (LC-CoA). Endocannabinoids are a class of lipids that are naturally expressed in a variety of cells, including cardiac, neuronal, and immune cells. While these lipids are identified as ligands for cannabinoid receptors there is a growing body of evidence that they can directly regulate the function of numerous ion channels independently of CBRs. Here we examine the effects of a panel of endocannabinoids on Kir2.1 function and demonstrate that a subset of endocannabinoids can alter Kir2.1 conductance to varying degrees independently of CBRs. Using computational and Surface plasmon resonance analysis, endocannabinoid regulation of Kir2.1 channels appears to be the result of altered membrane properties, rather than through direct protein-lipid interactions. Furthermore, differences in endocannabinoid effects on Kir4.1 and Kir7.1 channels, indicating that endocannabinoid regulation is not conserved among Kir family members. These findings may have broader implications on the function of cardiac, neuronal and/or immune cells.

Endothelium Piezo1 deletion alleviates experimental varicose veins by attenuating perivenous inflammation.

Previous large-scale genetic studies have prioritized the causal genes piezo type mechanosensitive ion channel component 1 (PIEZO1) and castor zinc finger 1 (CASZ1) associated with varicose veins (VVs). This study aims to evaluate their roles in both clinical and experimental VVs. In this study, we investigated abundance of PIEZO1 and CASZ1 in both varicose and normal veins from the same patients. Yoda1 (a selective PIEZO1 agonist, 2.6 mg/kg/day) or vehicle was administered intraperitoneally for 3 weeks to evaluate the effect of PIEZO1 activation on experimental VVs. Subsequently, endothelial Piezo1 deletion mice (Piezo1iΔEC mice) were generated to explored the role of endothelial PIEZO1 on VVs. Laser speckle imaging, flow cytometry, cell tracing with Evans blue or rhodamine-6G, and histopathological staining were utilized to evaluate the pathophysiology of VVs. Our results showed that mRNA expression of PIEZO1, but not CASZ1, was abundant and increased in clinical VVs. The Piezo1tP1-td mice revealed endothelium-specific expression of PIEZO1 in mice veins. By establishing iliac vein ligation-induced VVs in mice, Yoda1 exacerbated experimental VVs with increased inflammatory cell infiltration. Subsequently, endothelial Piezo1 deletion (Piezo1iΔEC mice) alleviated experimental VVs and vascular remodeling by directly reducing vascular permeability and leukocyte-endothelium interactions compared to the control (Piezo1fl/fl mice). PIEZO1 is highly expressed in clinical VVs, meanwhile, activation or inhibition of PIEZO1 exerts a remarkable effect on experimental VVs. Furthermore, Piezo1 may constitute a potential therapeutic approach for the medical treatment of VVs.

Structural prediction of GluN3 NMDA receptors.

N-methyl-D-aspartate (NMDA) receptors are heterotetrametric ion channels composed of two obligatory GluN1 subunits and two alternative GluN2 or GluN3 subunits, forming GluN1-N2, GluN1-N3, and GluN1-N2-N3 type of NMDA receptors. Extensive research has focused on the functional and structural properties of conventional GluN1-GluN2 NMDA receptors due to their early discovery and high expression levels. However, the knowledge of unconventional GluN1-N3 NMDA receptors remains limited. In this study, we modeled the GluN1-N3A, GluN1-N3B, and GluN1-N3A-N3B NMDA receptors using deep-learned protein-language predication algorithms AlphaFold and RoseTTAFold All-Atom. We then compared these structures with GluN1-N2 and GluN1-N3A receptor cryo-EM structures and found that GluN1-N3 receptors have distinct properties in subunit arrangement, domain swap, and domain interaction. Furthermore, we predicted the agonist- or antagonist-bound structures, highlighting the key molecular-residue interactions. Our findings shed new light on the structural and functional diversity of NMDA receptors and provide a new direction for drug development. This study uses advanced AI algorithms to model GluN1-N3 NMDA receptors, revealing unique structural properties and interactions compared to conventional GluN1-N2 receptors. By highlighting key molecular-residue interactions and predicting ligand-bound structures, our research enhances the understanding of NMDA receptor diversity and offers new insights for targeted drug development.

Design, synthesis, and in vitro biological evaluation of meta-sulfonamidobenzamide-based antibacterial LpxH inhibitors.

New antibacterial compounds are urgently needed, especially for infections caused by the top-priority Gram-negative bacteria that are increasingly difficult to treat. Lipid A is a key component of the Gram-negative outer membrane and the LpxH enzyme plays an important role in its biosynthesis, making it a promising antibacterial target. Inspired by previously reported ortho-N-methyl-sulfonamidobenzamide-based LpxH inhibitors, novel benzamide substitutions were explored in this work to assess their in vitro activity. Our findings reveal that maintaining wild-type antibacterial activity necessitates removal of the N-methyl group when shifting the ortho-N-methyl-sulfonamide to the meta-position. This discovery led to the synthesis of meta-sulfonamidobenzamide analogs with potent antibacterial activity and enzyme inhibition. Moreover, we demonstrate that modifying the benzamide scaffold can alter blocking of the cardiac voltage-gated potassium ion channel hERG. Furthermore, two LpxH-bound X-ray structures show how the enzyme-ligand interactions of the meta-sulfonamidobenzamide analogs differ from those of the previously reported ortho analogs. Overall, our study has identified meta-sulfonamidobenzamide derivatives as promising LpxH inhibitors with the potential for optimization in future antibacterial hit-to-lead programs.

Computational investigation in inhibitory effects of amantadine on classical swine fever virus p7 ion channel activity.

Classical swine fever virus (CSFV) p7 viroporin plays crucial roles in cellular ion balance and permeabilization. The antiviral drug amantadine effectively inhibits viral replication by blocking the activity of CSFV p7 viroporin. However, little information is available for the binding mode of amantadine with CSFV p7 viroporin, due to the lack of a known polymer structure for CSFV p7. In this study, we employed AlphaFold2 to predict CSFV p7 structures. Subsequently, we conducted a docking study to investigate the binding sites of amantadine to CSFV p7. Computational analysis showed that CSFV p7 forms a pore channel in a hexameric structure. Furthermore, molecular dynamics (MD) simulations and mutant analyses further suggest that CSFV p7 likely exists as a hexamer. Docking studies and MD simulations showed that amantadine interacts with the hydrophibic regions of tetramer and pentamer, as well as with the hydrophobic pore channel of the hexamer. Considering the potential hexameric assembly of CSFV p7, along with docking results, MD simulations, and the characteristics of the gated ion channels, we propose a model of CSFV p7 ion channel based on its hexameric configuration. In this model, residues E21, Y25, and R34 are suggested to selectively recruit and dehydrate ions, while residues L28 and L31 likely act as hydrophobic constrictors, thereby restricting the free movement of water. The binding of amantadine to residues I20, E21, V24 and Y25 effectively blocks ion transport. However, this proposed molecular model requires experimental validation. Our findings give a structural insight into the models of CSFV p7 as an ion channel and provide a molecular explanation for the inhibition effects of amantadine on CSFV p7-mediated ion channel conductance.

Progress of the Impact of Terahertz Radiation on Ion Channel Kinetics in Neuronal Cells.

In neurons and myocytes, selective ion channels in the plasma membrane play a pivotal role in transducing chemical or sensory stimuli into electrical signals, underpinning neural and cardiac functionality. Recent advancements in biomedical research have increasingly spotlighted the interaction between ion channels and electromagnetic fields, especially terahertz (THz) radiation. This review synthesizes current findings on the impact of THz radiation, known for its deep penetration and non-ionizing properties, on ion channel kinetics and membrane fluid dynamics. It is organized into three parts: the biophysical effects of THz exposure on cells, the specific modulation of ion channels by THz radiation, and the potential pathophysiological consequences of THz exposure. Understanding the biophysical mechanisms underlying these effects could lead to new therapeutic strategies for diseases.

Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM.

Ryanodine receptors (RyRs) are large Ca2+ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within four hours on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited.

Crosstalk between PKA and PIAS3 regulates cardiac Kv4 channel SUMOylation.

Post-translational SUMOylation of nuclear and cytosolic proteins maintains homeostasis in eukaryotic cells and orchestrates programmed responses to changes in metabolic demand or extracellular stimuli. In excitable cells, SUMOylation tunes the biophysical properties and trafficking of ion channels. Ion channel SUMOylation status is determined by the opposing enzyme activities of SUMO ligases and deconjugases. Phosphorylation also plays a permissive role in SUMOylation. SUMO deconjugases have been identified for several ion channels, but their corresponding E3 ligases remain unknown. This study shows PIAS3, a.k.a. KChAP, is a bona fide SUMO E3 ligase for Kv4.2 and HCN2 channels in HEK cells, and endogenous Kv4.2 and Kv4.3 channels in cardiomyocytes. PIAS3-mediated SUMOylation at Kv4.2-K579 increases channel surface expression through a rab11a-dependent recycling mechanism. PKA phosphorylation at Kv4.2-S552 reduces the current mediated by Kv4 channels in HEK293 cells, cardiomyocytes, and neurons. This study shows PKA mediated phosphorylation blocks Kv4.2-K579 SUMOylation in HEK cells and cardiomyocytes. Together, these data identify PIAS3 as a key downstream mediator in signaling cascades that control ion channel surface expression.

Structure-function and pharmacologic aspects of ion channels relevant to neurologic channelopathies.

Ion channels are membrane proteins that allow the passage of ions across the membrane. They characteristically contain a pore where the selectivity of certain ion species is determined and gates that open and close the pore are found. The pore is often connected to additional domains or subunits that regulate its function. Channels are grouped into families based on their selectivity for specific ions and the stimuli that control channel opening and closing, such as voltage or ligands. Ion channels are fundamental to the electrical properties of excitable tissues. Dysfunction of channels can lead to abnormal electrical signaling of neurons and muscle cells, accompanied by clinical manifestations, known as channelopathies. Many naturally occurring toxins target ion channels and affect excitable cells where the channels are expressed. Furthermore, ion channels, as membrane proteins and key regulators of a number of physiologic functions, are an important target for drugs in clinical use. In this chapter, we give a general overview of the classification, genetics and structure-function features of the main ion channel families, and address some pharmacologic aspects relevant to neurologic channelopathies.

Inherited myotonias.

The inherited myotonias are a complex group of diseases caused by variations in genes that encode or modulate the expression of ion channels that regulate muscle excitability. These variations alter muscle membrane excitability allowing mild depolarization, causing myotonic discharges. There are two groups of inherited myotonia, the dystrophic and the nondystrophic myotonias (NDM). Patients with NDM have a pure muscle phenotype with variations in channel genes expressed in muscle. The dystrophic myotonias are caused by genes that alter splicing leading to more systemic effects with myotonia being one of a number of systemic symptoms. This chapter therefore focuses on the key aspects of the NDMs. The NDMs manifest with varying clinical phenotypes, which change from infancy to adulthood. The pathogenicity of different variants can be determined using heterologous expression systems to understand the alteration in channel properties and predict the likelihood of causing disease. Myotonia itself can be managed by lifestyle modifications. A number of randomized controlled trials demonstrate efficacy of mexiletine and lamotrigine in treating myotonia, but there is an evidence that specific variants may be more or less well-treated by the different agents because of how they alter the channel kinetics. More work is needed to develop more targeted genetic treatments.

Reviewing mathematical models of sperm signaling networks.

Dave Garbers' work significantly contributed to our understanding of sperm's regulated motility, capacitation, and the acrosome reaction. These key sperm functions involve complex multistep signaling pathways engaging numerous finely orchestrated elements. Despite significant progress, many parameters and interactions among these elements remain elusive. Mathematical modeling emerges as a potent tool to study sperm physiology, providing a framework to integrate experimental results and capture functional dynamics considering biochemical, biophysical, and cellular elements. Depending on research objectives, different modeling strategies, broadly categorized into continuous and discrete approaches, reveal valuable insights into cell function. These models allow the exploration of hypotheses regarding molecules, conditions, and pathways, whenever they become challenging to evaluate experimentally. This review presents an overview of current theoretical and experimental efforts to understand sperm motility regulation, capacitation, and the acrosome reaction. We discuss the strengths and weaknesses of different modeling strategies and highlight key findings and unresolved questions. Notable discoveries include the importance of specific ion channels, the role of intracellular molecular heterogeneity in capacitation and the acrosome reaction, and the impact of pH changes on acrosomal exocytosis. Ultimately, this review underscores the crucial importance of mathematical frameworks in advancing our understanding of sperm physiology and guiding future experimental investigations.

A nondestructive membrane engineering method using an amphiphilic polymer.

The cellular signaling process or ion transport is mediated by membrane proteins (MPs) located on the cell surface, and functional studies of MPs have mainly been conducted using cells endogenously or transiently expressing target proteins. Reconstitution of purified MPs in the surface of live cells would have advantages of short manipulation time and ability to target cells in which gene transfection is difficult. However, direct reconstitution of MPs in live cells has not been established. The traditional detergent-mediated reconstitution method of MPs into a lipid bilayer cannot be applied to live cells because this disrupts and reforms the lipid bilayer structure, which is detrimental to cell viability. In this study, we demonstrated that GPCRs (prostaglandin E2 receptor 4 [EP4] and glucagon-like peptide-1 receptor [GLP1R]) or serotonin receptor 3A (5HT3A), a ligand-gated ion channel, stabilized with amphiphilic poly-γ-glutamate (APG), can be reconstituted into mammalian cell plasma membranes without affecting cell viability. Furthermore, 5HT3A reconstituted in mammalian cells showed ligand-dependent Ca2+ ion transport activity. APG-mediated reconstitution of GPCR in synthetic liposomes showed that electrostatic interaction between APG and membrane surface charge contributed to the reconstitution process. This APG-mediated membrane engineering method could be applied to the functional modification of cell membranes with MPs in live cells.

Protein Kinase A Inhibitor Attenuates the Antinociceptive Effect of NMDA-Receptor Channel Antagonists in the Capsaicin Test in Mice.

Acute nociceptive pain in mice caused by subcutaneous (intraplantar) injection of TRPV1 ion channel agonist capsaicin (1.6 µg/mouse) and the effects of protein kinase A inhibitor H-89 (0.05 mg/mouse, intraplantar injection) and NMDA receptor channel antagonists MK-801 (7.5 and 15 µg/mouse, topical application) and hemantane (0.5 mg/mouse, topical application) on the pain were assessed. MK-801 and hemantane were found to reduce the duration of the pain response. H-89 did not significantly affect the pain in animals, but preliminary administration of this drug abolished the antinociceptive effect of MK-801 (7.5 µg/mouse) and weakens the effect of hemantane (0.5 mg/mouse).

The odyssey of the TR(i)P journey to the cellular membrane.

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.

Unlocking cellular traffic jams: olive oil-mediated rescue of CNG mutant channels.

One of the reasons to suggest olive oil consumption for a healthy life is its potential to induce robust lipidomic remodeling through membrane modification by dietary lipids. This remodeling might, in turn, modulate essential lipid-protein interactions while maintaining accurate transmembrane protein/domain orientation. Oleic acid, the primary compound in olive oil, has been suggested as a modulator of ion channel function. In this study, we explored whether this lipid could rescue the trafficking of mutated transmembrane proteins. In our initial approach, we supplemented the cell culture medium of HEK-293 cells expressing cyclic nucleotide channels tagged using green fluorescent protein (CNG-GFP) with olive oil or oleic acid. In addition to wild-type channels, we also expressed R272Q and R278W mutant channels, two non-functional intracellularly retained channels related to retinopathies. We used fluorescence microscopy and patch-clamp in the inside-out configuration to assess changes in the cell localization and function of the tested channels. Our results demonstrated that olive oil and oleic acid facilitated the transport of cyclic nucleotide-gated R272Q mutant channels towards the plasma membrane, rendering them electrophysiologically functional. Thus, our findings reveal a novel property of olive oil as a membrane protein traffic inductor.

Activation of the influenza B M2 proton channel (BM2).

Influenza B viruses have co-circulated during most seasonal flu epidemics and can cause significant human morbidity and mortality due to their rapid mutation, emerging drug resistance, and severe impact on vulnerable populations. The influenza B M2 proton channel (BM2) plays an essential role in viral replication, but the mechanisms behind its symmetric proton conductance and the involvement of a second histidine (His27) cluster remain unclear. Here we perform the membrane-enabled continuous constant-pH molecular dynamics simulations on wildtype BM2 and a key H27A mutant to explore its pH-dependent conformational switch. Simulations capture the activation as the first histidine (His19) protonates and reveal the transition at lower pH values compared to AM2 is a result of electrostatic repulsions between His19 and pre-protonated His27. Crucially, we provide an atomic-level understanding of the symmetric proton conduction by identifying pre-activating channel hydration in the C-terminal portion. This research advances our understanding of the function of BM2 function and lays the groundwork for further chemically reactive modeling of the explicit proton transport process as well as possible anti-flu drug design efforts.

Scorpion α-toxin LqhαIT specifically interacts with a glycan at the pore domain of voltage-gated sodium channels.

Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. To elucidate the mechanism of action of LqhαIT, we determined a 3.9 Å cryoelectron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in an "S4 down" conformation. The functionally essential C-terminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrates the functional importance of this toxin-glycan interaction. These findings establish a structural basis for the specificity achieved by scorpion α-toxins and reveal the conserved glycan as an essential component of the toxin-binding epitope.

The Phytochemical, Quercetin, Attenuates Nociceptive and Pathological Pain: Neurophysiological Mechanisms and Therapeutic Potential.

Although phytochemicals are plant-derived toxins that are primarily produced as a form of defense against insects or microbes, several lines of study have demonstrated that the phytochemical, quercetin, has several beneficial biological actions for human health, including antioxidant and inflammatory effects without side effects. Quercetin is a flavonoid that is widely found in fruits and vegetables. Since recent studies have demonstrated that quercetin can modulate neuronal excitability in the nervous system, including nociceptive sensory transmission via mechanoreceptors and voltage-gated ion channels, and inhibit the cyclooxygenase-2-cascade, it is possible that quercetin could be a complementary alternative medicine candidate; specifically, a therapeutic agent against nociceptive and pathological pain. The focus of this review is to elucidate the neurophysiological mechanisms underlying the modulatory effects of quercetin on nociceptive neuronal activity under nociceptive and pathological conditions, without inducing side effects. Based on the results of our previous research on trigeminal pain, we have confirmed in vivo that the phytochemical, quercetin, demonstrates (i) a local anesthetic effect on nociceptive pain, (ii) a local anesthetic effect on pain related to acute inflammation, and (iii) an anti-inflammatory effect on chronic pain. In addition, we discuss the contribution of quercetin to the relief of nociceptive and inflammatory pain and its potential clinical application.

A BTB extension and ion-binding domain contribute to the pentameric structure and TFAP2A binding of KCTD1.

KCTD family proteins typically assemble into cullin-RING E3 ligases. KCTD1 is an atypical member that functions instead as a transcriptional repressor. Mutations in KCTD1 cause developmental abnormalities and kidney fibrosis in scalp-ear-nipple syndrome. Here, we present unexpected mechanistic insights from the structure of human KCTD1. Disease-causing mutation P20S maps to an unrecognized extension of the BTB domain that contributes to both its pentameric structure and TFAP2A binding. The C-terminal domain (CTD) shares its fold and pentameric assembly with the GTP cyclohydrolase I feedback regulatory protein (GFRP) despite lacking discernible sequence similarity. Most surprisingly, the KCTD1 CTD establishes a central channel occupied by alternating sodium and iodide ions that restrict TFAP2A dissociation. The elucidation of the structure redefines the KCTD1 BTB domain fold and identifies an unexpected ion-binding site for future study of KCTD1's function in the ectoderm, neural crest, and kidney.

Differential expression of ion channel coding genes in the endometrium of women experiencing recurrent implantation failures.

Our study probed the differences in ion channel gene expression in the endometrium of women with Recurrent Implantation Failure (RIF) compared to fertile women. We analyzed the relative expression of genes coding for T-type Ca2+, ENaC, CFTR, and KCNQ1 channels in endometrial samples from 20 RIF-affected and 10 control women, aged 22-35, via microarray analysis and quantitative real-time PCR. Additionally, we examined DNA methylation in the regulatory region of KCNQ1 using ChIP real-time PCR. The bioinformatics component of our research included Gene Ontology analysis, protein-protein interaction networks, and signaling pathway mapping to identify key biological processes and pathways implicated in RIF. This led to the discovery of significant alterations in the expression of ion channel genes in RIF women's endometrium, most notably an overexpression of CFTR and reduced expression of SCNN1A, SCNN1B, SCNN1G, CACNA1H, and KCNQ1. A higher DNA methylation level of KCNQ1's regulatory region was also observed in RIF patients. Gene-set enrichment analysis highlighted a significant presence of genes involved with ion transport and membrane potential regulation, particularly in sodium and calcium channel complexes, which are vital for cation movement across cell membranes. Genes were also enriched in broader ion channel and transmembrane transporter complexes, underscoring their potential extensive role in cellular ion homeostasis and signaling. These findings suggest a potential involvement of ion channels in the pathology of implantation failure, offering new insights into the mechanisms behind RIF and possible therapeutic targets.