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.

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).

Insulin enhances acid-sensing ion channel currents in rat primary sensory neurons.

Insulin has been shown to modulate neuronal processes through insulin receptors. The ion channels located on neurons may be important targets for insulin/insulin receptor signaling. Both insulin receptors and acid-sensing ion channels (ASICs) are expressed in dorsal root ganglia (DRG) neurons. However, it is still unclear whether there is an interaction between them. Therefore, the purpose of this investigation was to determine the effects of insulin on the functional activity of ASICs. A 5 min application of insulin rapidly enhanced acid-evoked ASIC currents in rat DRG neurons in a concentration-dependent manner. Insulin shifted the concentration-response plot for ASIC currents upward, with an increase of 46.2 ± 7.6% in the maximal current response. The insulin-induced increase in ASIC currents was eliminated by the insulin receptor antagonist GSK1838705, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol-3 kinase antagonist wortmannin. Moreover, insulin increased the number of acid-triggered action potentials by activating insulin receptors. Finally, local administration of insulin exacerbated the spontaneous nociceptive behaviors induced by intraplantar acid injection and the mechanical hyperalgesia induced by intramuscular acid injections through peripheral insulin receptors. These results suggested that insulin/insulin receptor signaling enhanced the functional activity of ASICs via tyrosine kinase and phosphatidylinositol-3 kinase pathways. Our findings revealed that ASICs were targets in primary sensory neurons for insulin receptor signaling, which may underlie insulin modulation of pain.

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.

Ursolic acid, an inhibitor of TMEM16A, co-loaded with cisplatin in hydrogel drug delivery system for multi-targeted therapy of lung cancer.

The efficacy of single chemotherapy drugs in cancer treatment is often limited. Combining administration targeting multiple targets has emerged as an effective strategy to improve cancer treatment. Ursolic acid, a triterpenoid compound in various natural foods, was identified as a novel inhibitor of lung cancer specific target TMEM16A. The IC50 of ursolic acid on the whole-cell current of TMEM16A was 13.85 ± 1.64 µM. Molecular dynamics simulations and site-directed mutagenesis experiments indicated the binding sites of ursolic acid on TMEM16A as L381, R535, E623, and C625. Ursolic acid significantly inhibited the proliferation and migration of LA795 cells, while promoting cancer cell apoptosis. Mechanistic studies revealed that ursolic acid inhibited lung cancer through the MAPK and EMT pathways, and induced DNA and membrane damage. Next, a degradable and self-repairing hydrogel drug-loading system was designed to enhance the targeting effect of the ursolic acid and cisplatin drug combination. In vivo experiments showed that the hydrogel-loaded ursolic acid and cisplatin enhanced the antitumor activity and reduced the toxicity. This study presents a novel approach of multi-target combination therapy using ursolic acid and cisplatin, combined with the targeted delivery capability of the hydrogel system, which significantly improves the therapeutic efficacy in lung cancer.

Delivery of siRNAs Against Selective Ion Channels and Transporter Genes Using Hyaluronic Acid-coupled Carbonate Apatite Nanoparticles Synergistically Inhibits Growth and Survival of Breast Cancer Cells.

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.

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.

Unraveling the molecular reason of opposing effects of α-mangostin and norfluoxetine on TREK-2 at the same binding site [[EQUATION]].

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.

Using Nanoscale Passports To Understand and Unlock Ion Channels as Gatekeepers of the Cell.

Natural ion channels are proteins embedded in the cell membrane that control many aspects of cell and human physiology by acting as gatekeepers, regulating the flow of ions in and out of cells. Advances in nanotechnology have influenced the methods for studying ion channels in vitro, as well as ways to unlock the delivery of therapeutics by modulating them in vivo. This review provides an overview of nanotechnology-enabled approaches for ion channel research with a focus on the synthesis and applications of synthetic ion channels. Further, the uses of nanotechnology for therapeutic applications are critically analyzed. Finally, we provide an outlook on the opportunities and challenges at the intersection of nanotechnology and ion channels. This work highlights the key role of nanoscale interactions in the operation and modulation of ion channels, which may prompt insights into nanotechnology-enabled mechanisms to study and exploit these systems in the near future.

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.

Insights into the antifungal mechanism of Bacillus subtilis cyclic lipopeptide iturin A mediated by potassium ion channel.

Fungal infections pose severe and potentially lethal threats to plant, animal, and human health. Ergosterol has served as the primary target for developing antifungal medications. However, many antifungal drugs remain highly toxic to humans due to similarity in cell membrane composition between fungal and animal cells. Iturin A, lipopeptide produced by Bacillus subtilis, efficiently inhibit various fungi, but demonstrated safety in oral administration, indicating the existence of targets different from ergosterol. To pinpoint the exact antifungal target of iturin A, we used homologous recombination to knock out and overexpress erg3, a key gene in ergosterol synthesis. Saccharomyces cerevisiae and Aspergillus carbonarius were transformed using the LiAc/SS-DNNPEG and Agrobacterium-mediated transformation (AMT), respectively. Surprisingly, increasing ergosterol content did not augment antifungal activity. Furthermore, iturin A's antifungal activity against S. cerevisiae was reduced while it pre-incubation with voltage-gated potassium (Kv) channel inhibitor, indicating that Kv activation was responsible for cell death. Iturin A was found to activate the Kv protein, stimulating K+ efflux from cell. In vitro tests confirmed interaction between iturin A and Kv protein. This study highlights Kv as one of the precise targets of iturin A in its antifungal activity, offering a novel target for the development of antifungal medications.

Novel interplay between agonist and calcium binding sites modulates drug potentiation of α7 acetylcholine receptor.

Drug modulation of the α7 acetylcholine receptor has emerged as a therapeutic strategy for neurological, neurodegenerative, and inflammatory disorders. α7 is a homo-pentamer containing topographically distinct sites for agonists, calcium, and drug modulators with each type of site present in five copies. However, functional relationships between agonist, calcium, and drug modulator sites remain poorly understood. To investigate these relationships, we manipulated the number of agonist binding sites, and monitored potentiation of ACh-elicited single-channel currents through α7 receptors by PNU-120596 (PNU) both in the presence and absence of calcium. When ACh is present alone, it elicits brief, sub-millisecond channel openings, however when ACh is present with PNU it elicits long clusters of potentiated openings. In receptors harboring five agonist binding sites, PNU potentiates regardless of the presence or absence of calcium, whereas in receptors harboring one agonist binding site, PNU potentiates in the presence but not the absence of calcium. By varying the numbers of agonist and calcium binding sites we show that PNU potentiation of α7 depends on a balance between agonist occupancy of the orthosteric sites and calcium occupancy of the allosteric sites. The findings suggest that in the local cellular environment, fluctuations in the concentrations of neurotransmitter and calcium may alter this balance and modulate the ability of PNU to potentiate α7.

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.

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.

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.

A frozen portrait of a warm channel.

In order to understand protein function, the field of structural biology makes extensive use of cryogenic electron microscopy (cryo-EM), a technique that enables structure determination at atomic resolution following embedding of protein particles in vitreous ice. Considering the profound effects of temperature on macromolecule function, an important-but often neglected-question is how the frozen particles relate to the actual protein conformations at physiological temperatures. In a recent study, Hu et al. compare structures of the cation channel TRPM4 "frozen" at 4 °C versus 37 °C, revealing how temperature critically affects the binding of activating Ca2+ ions and other channel modulators.

CXCL10 Enhances Acid-Sensing Ion Channel Currents in Rat Dorsal Root.

Both CXCL10/CXCR3 and acid-sensing ion channels (ASICs) are expressed in nociceptive sensory neurons and participate in various pain processes, but it is still unclear whether there is a link between them. Herein, we report that CXCL10 enhances the electrophysiological activity of ASICs in rat dorsal root ganglia (DRG) neurons. A brief (10 min) application of CXCL10 increased acid-evoked ASIC currents in a concentration-dependent manner. CXCL10 increased the maximum response of ASICs to acidic stimuli without changing their sensitivity. CXCL10 enhanced ASIC currents in DRG cells through CXCR3, as this enhancement was completely blocked by AMG487, a selective CXCR3 antagonist. CXCL10 also increased ASIC3 currents in CHO cells coexpressing ASIC3 and CXCR3 but not in cells expressing ASIC3 alone. The CXCL10-mediated increase in ASIC currents was prevented by the application of either the G protein inhibitor GDP-ß-S or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 but not by the ERK inhibitor U0126 or the JNK inhibitor SP600125. Moreover, CXCL10 increased the number of action potentials triggered by acidic stimuli via CXCR3. CXCL10 dose-dependently exacerbated acid-induced nociceptive behavior in rats through peripheral CXCR3. These results indicated that CXCL10/CXCR3 signaling enhanced ASIC-mediated electrophysiological activity in DRG neurons and nociception in rats via a p38 MAPK-dependent pathway, revealing a novel mechanism underlying pain. CXCL10/CXCR3 signaling may be an effective target in the treatment of pain associated with tissue acidification.