RESUMEN

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.

RESUMEN

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.

RESUMEN

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.

Asunto(s)

RESUMEN

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.

RESUMEN

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.

RESUMEN

Transient receptor potential (TRP) ion channels serve as sensors for variations in ambient temperature, modulating both thermoregulation and temperature responsive cellular processes. Among these, the vanilloid TRP subfamily (TRPV) comprises six members and at least four of these members (TRPV1-TRPV4) have been associated with thermal sensation. TRPV2 has been described as a sensor for noxious heat, but subsequent studies have unveiled a more complex role for TRPV2 beyond temperature perception. This comprehensive review aims to elucidate the intricate thermosensitivity of TRPV2 by synthesizing current knowledge on its biophysical properties, expression pattern and known physiological functions associated with thermosensation.

Asunto(s)

RESUMEN

Ultrasound is a mechanical wave that can non-invasively penetrate the skull to deep brain regions to activate neurons. Transcranial focused ultrasound neuromodulation is a promising approach, with the advantages of noninvasiveness, high-resolution, and deep penetration, which developed rapidly over the past years. However, conventional transcranial ultrasound's spatial resolution is low-precision which hinders its use in precision neuromodulation. Here we focus on methods that could increase the spatial resolution, gain modulation efficiency at the focal spot, and potential mechanisms of ultrasound neuromodulation. In this paper, we summarize strategies to enhance the precision of ultrasound stimulation, which could potentially improve the ultrasound neuromodulation technic.

RESUMEN

A quintessential sentinel of cell health, the membrane potential in nonexcitable cells integrates biochemical and biomechanical inputs, determines the driving force for ionic currents activated by input signals and plays critical functions in cellular differentiation, signaling, and pathology. The identity and properties of ion channels that subserve the resting potential in trabecular meshwork (TM) cells is poorly understood, which impairs our understanding of intraocular pressure regulation in healthy and diseased eyes. Here, we identified a powerful cationic conductance that subserves the TM resting potential. It disappears following Na+ removal or substitution with choline or NMDG+, is insensitive to TTX, verapamil, phenamil methanesulfonate, amiloride and GsMTx4, is substituted by Li+ and Cs+, and inhibited by Gd3+ and Ruthenium Red. Constitutive cation influx is thus not mediated by voltage-operated Na+, Ca2+, epithelial Na+ (ENaC) channels, Piezo channels or Na+/H+ exchange but may involve TRP-like channels. Transcriptional analysis detected expression of many TRP genes, with the transcriptome pool dominated by TRPC1 followed by expression of TRPV1, TRPC3, TRPV4 and TRPC5. Pyr3 and Pico1,4,5 did not affect the standing current whereas SKF96365 promoted rather than suppressed, Na+ influx. SEA-0400 induced a modest hyperpolarization, indicating residual contribution from Na+/Ca2+ exchange. The resting membrane potential in human TM cells is thus maintained by a constitutive monovalent cation leak current with properties not unlike those of TRP channels. This conductance is likely to influence conventional outflow by setting the homeostatic steady-state and by regulating the magnitude of pressure-induced currents in normotensive and hypertensive eyes.

RESUMEN

Cancer remains a complex and multifaceted disease, characterized by a myriad of molecular and cellular alterations that collectively drive tumorigenesis and progression. Hanahan and Weinberg's concept of cancer hallmarks has offered a framework for comprehending the various but related aspects of cancer biology. Initially defined as a set of six hallmarks, further investigation has added more characteristics to this list that also contribute to the malignant phenotype. Changes in cellular energetics, proliferative signaling, and resistance to cell death are three of these hallmarks that have been thoroughly investigated and described. But new discoveries in the field of cancer biology have brought attention to the importance of another aspect of the biology of cancer: the dysregulation of membrane potential.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

Alterations in anion balance potential, along with the involvement of cation-chloride cotransporters, play pivotal roles in the development of hyperalgesia after peripheral nerve injury (PNI). Chloride voltage-gated channel 7 (CLCN7) is the predominant member of the CLC protein family. Investigations on CLCN7 have focused primarily on its involvement in osteosclerosis and lysosomal storage disorders; nevertheless, its contribution to neuropathic pain (NP) has not been determined. In this investigation, we noted high expression of CLCN7 in neurons situated within the spinal dorsal horns (SDHs) and dorsal root ganglions (DRGs). Immunofluorescence analysis revealed that CLCN7 was predominantly distributed among IB4-positive and CGRP-positive neurons. Furthermore, the expression of CLCN7 was observed to be mainly reduced in neurons within the SDHs and in small and medium-sized neurons located in the DRGs of spared nerve injury (SNI) mice. Knockdown of CLCN7 via siRNA in the DRGs resulted in increased mechanical and thermal hyperalgesia in naïve mice. Furthermore, the excitability of cultured DRG neurons in vitro was augmented upon treatment with CLCN7 siRNA. These findings suggested that CLCN7 downregulation following SNI was crucial for the manifestation of mechanical and thermal hyperalgesia, highlighting potential targeting strategies for treating NP.

RESUMEN

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.

Asunto(s)

RESUMEN

Background: Acid-sensing ion channel 1a (ASIC1a) plays a critical role in physiological and pathological processes. To further elucidate the biological functions of ASICs and their relationships with disease occurrence and development, it is advantageous to investigate and develop additional regulatory factors for ASICs. Methods: In this study, cation exchange chromatography was used to separate seven chromatographic components from Naja naja atra venom. Capillary electrophoresis was employed to detect that Ⅶ peak component containing a main protein Ⅶ-2, which could bind to ASIC1a. The analgesic effects of Ⅶ-2 protein were determined using hot plate methods, and ASIC1a expression in spinal cord tissue from rats with inflammatory pain was detected using western blot. Results: The purified Ⅶ-2 protein named Naja naja atra venom-Ⅶ-2 (NNAV-Ⅶ-2) was obtained by Sephadex G-50 gel filtration, which exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 6.7 kD. Remarkably, the NNAV-Ⅶ-2 protein demonstrated a significant analgesic effect and downregulated ASIC1a expression in the spinal cord tissue of rats with inflammatory pain. Conclusions: The analgesic mechanism of the NNAV-Ⅶ-2 protein may be associated with its binding to ASIC1a, consequently downregulating ASIC1a expression in neural tissues.

RESUMEN

Voltage-gated proton channel (Hv) has activity of proton transport following electrochemical gradient of proton. Hv is expressed in neutrophils and macrophages of which functions are physiologically temperature-sensitive. Hv is also expressed in human sperm cells and regulates their locomotion. H+ transport through Hv is both regulated by membrane potential and pH difference across biological membrane. It is also reported that properties of Hv such as proton conductance and gating are highly temperature-dependent. Hv consists of the N-terminal cytoplasmic domain, the voltage sensor domain (VSD), and the C-terminal coiled-coil domain, and H+ permeates through VSD voltage-dependently. The functional unit of Hv is a dimer via the interaction between C-terminal coiled-coils assembly domain. We have reported that the coiled-coil domain of Hv has the nature of dissociation around our bodily temperature and mutational change of the coiled-coil affected temperature-sensitive gating, especially its temperature threshold. The temperature-sensitive gating is assessed from two separate points: temperature threshold and temperature dependence. In this chapter, I describe physiological roles and molecular structure mechanisms of Hv by mainly focusing on thermosensitive properties.

Asunto(s)

RESUMEN

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.

RESUMEN

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).

Asunto(s)

RESUMEN

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.

RESUMEN

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.

RESUMEN

Introduction: Dysregulated calcium homeostasis and consequentially aberrant Ca2+ signalling could enhance survival, proliferation and metastasis in various cancers. Despite rapid development in exploring the ion channel functions in relation to cancer, most of the mechanisms accounting for the impact of ion channel modulators have yet to be fully clarified. Although harnessing small interfering RNA (siRNA) to specifically silence gene expression has the potential to be a pivotal approach, its success in therapeutic intervention is dependent on an efficient delivery system. Nanoparticles have the capacity to strongly bind siRNAs. They remain in the circulation and eventually deliver the siRNA payload to the target organ. Afterward, they interact with the cell surface and enter the cell via endocytosis. Finally, they help escape the endo-lysosomal degradation system prior to unload the siRNAs into cytosol. Carbonate apatite (CA) nanocrystals primarily is composed of Ca2+, carbonate and phosphate. CA possesses both anion and cation binding domains to target negatively charged siRNA molecules. Methods: Hybrid CA was synthesized by complexing CA NPs with a hydrophilic polysaccharide - hyaluronic acid (HA). The average diameter of the composite particles was determined using Zetasizer and FE-SEM and their zeta potential values were also measured. Results and Discussion: The stronger binding affinity and cellular uptake of a fluorescent siRNA were observed for HA-CA NPs as compared to plain CA NPs. Hybrid CA was electrostatically bound individually and combined with three different siRNAs to silence expression of calcium ion channel and transporter genes, TRPC6, TRPM8 and SLC41A1 in a human breast cancer cell line (MCF-7) and evaluate their potential for treating breast cancer. Hybrid NPs carrying TRPC6, TRPM8 and SLC41A1 siRNAs could significantly enhance cytotoxicity both in vitro and in vivo. The resultant composite CA influenced biodistribution of the delivered siRNA, facilitating reduced off target distribution and enhanced breast tumor targetability.

Asunto(s)

RESUMEN

TWIK-related K(+) channel (TREK)-2, expressed in sensory neurons, is involved in setting membrane potential, and its modulations contributes to the generation of nociceptive signals. Although acute and chronic pain is a common symptom experienced by patients with various conditions, most existing analgesics exhibit low efficacy and are associated with adverse effects. For this reason, finding the novel modulator of TREK-2 is of significance for the development of new analgesics. Recent studies have shown that α-Mangostin (α-MG) activates TREK-2, facilitating analgesic effects, yet the underlying molecular mechanisms remain elusive. Intriguingly, even though norfluoxetine (NFx) is known to inhibit TREK-2, α-MG is also observed to share the same binding site with NFx, and this implies that TREK-2 might be modulated in a highly complicated manner. Therefore, we examine the mechanism of how TREK-2 is activated by α-MG using computational methods and patch clamp experiments in the present study. Based on these results, we offer an explanation of how α-MG and NFx exhibit opposing effects at the same binding site of TREK-2. These findings will broaden our understanding of TREK-2 modulation, providing clues for designing novel analgesic drugs.