Membrane Ion Channels Research Articles

Development of an Intravenously Stable Disulfide-Rich Peptide for the Treatment of Chemotherapy-Induced Neuropathic Pain.

α-conotoxins (α-Ctxs), a class of disulfide-rich conopetides, are excellent drug leads due to their small size, high selectivity, and potency for specific membrane receptors and ion channels involved in pain transmission. However, their high susceptibility to proteolytic degradation limits their therapeutic potential. In this study, we designed and synthesized a series of conformationally stable analogues of α-Ctx Mr1.1[S4Dap] using various structural optimization strategies. The Mr1.1[S4Dap, C16Pen] analogue maintained potency at human α9α10 nicotinic acetylcholine receptors, with a half-maximal inhibitory concentration (IC50) of 4 nM. It exhibited over a 5-fold increase in serum stability compared to Mr1.1[S4Dap], without disrupting its overall conformation. Furthermore, intravenous application of Mr1.1[S4Dap, C16Pen] showed potent analgesic activity in oxaliplatin-induced cold allodynia, indicating a high potential for drug development. Overall, the results from this study provide valuable insights for optimizing the serum stability of disulfide-rich peptides in future therapeutic applications.

Implications of TRPM3 and TRPM8 for sensory neuron sensitisation.

Sensory neurons serve to receive and transmit a wide range of information about the conditions of the world around us as well as the external and internal state of our body. Sensitisation of these nerve cells, i.e.becoming more sensitive to stimuli or the emergence or intensification of spontaneous activity, for example in the context of inflammation or nerve injury, can lead to chronic diseases such as neuropathic pain. For many of these disorders there are only very limited treatment options and in order to find and establish new therapeutic approaches, research into the exact causes of sensitisation with the elucidation ofthe underlying mechanisms and the identification of themolecular components is therefore essential. These components include plasma membrane receptors and ion channels that are involved in signal reception and transmission. Members of the transient receptor potential (TRP) channel family are also expressed in sensory neurons and some of them play a crucial role in temperature perception. This review article focuses on the heat-sensitive TRPM3 andthe cold-sensitive TRPM8 (and TRPA1) channels and their importance in sensitisation of dorsal root ganglion sensory neurons is discussed based on studies related to inflammation and injury- as well as chemotherapy-induced neuropathy.

Sudden Cardiac Death and Channelopathies: What Lies behind the Clinical Significance of Rare Splice-Site Alterations in the Genes Involved?

Background and objectives: Sudden cardiac death (SCD) is a natural and unexpected death of cardiac origin that occurs within 1 h from the onset of acute symptoms. The major leading causes of SCD are cardiomyopathies and channelopathies. In this review, we focus on channelopathies, inherited diseases caused by mutations affecting genes encoding membrane ion channels (sodium, potassium or calcium channels) or cellular structures that affect Ca2+ availability. The diagnosis of diseases such as long QT, Brugada syndrome, short QT and catecholaminergic polymorphic ventricular tachycardia (CPVT) is still challenging. Currently, genetic testing and next-generation sequencing allow us to identify many rare alterations. However, some non-coding variants, e.g., splice-site variants, are usually difficult to interpret and to classify. Methods: In our review, we searched for splice-site variants of genes involved in channelopathies, focusing on variants of unknown significance (VUSs) registered on ClinVar up to now. Results: The research led to a high number of splice-site VUSs of genes involved in channelopathies, suggesting the performance of deeper studies. Conclusions: In order to interpret the correlation between variants and pathologies, we discuss experimental studies, such as RNA sequencing and functional analysis of proteins. Unfortunately, as these in vitro analyses cannot always be performed, we draw attention to in silico studies as future perspectives in genetics. This review has the aim of discussing the potential methods of detection and interpretation of VUSs, bringing out the need for a future reclassification of variants with currently unknown significance.

Resting human trabecular meshwork cells experience tonic cation influx.

The trabecular meshwork (TM) regulates intraocular pressure (IOP) by converting biochemical and biomechanical stimuli into intracellular signals. Recent electrophysiological studies demonstrated that this process is mediated by pressure sensing ion channels in the TM plasma membrane while the molecular and functional properties of channels that underpin ionic homeostasis in resting cells remain largely unknown. Here, we demonstrate that the TM resting potential is subserved by a powerful cationic conductance that disappears following Na+ removal and substitution with choline or NMDG+. Its insensitivity to TTX, verapamil, phenamil methanesulfonate and amiloride indicates it does not involve voltage-operated Na+, Ca2+ and epithelial Na+ (ENaC) channels or Na+/H+ exchange while a modest hyperpolarization induced by SEA-0440 indicates residual contribution from reversed Na+/Ca2+ exchange. Tonic cationic influx was inhibited by Gd3+ and Ruthenium Red but not GsMTx4, indicating involvement of TRP-like but not Piezo channels. Transcriptional analysis detected expression of most TRP genes, with the canonical transcriptome pool dominated by TRPC1 followed by the expression ofTRPV1, TRPC3 and TRPC5. TRPC3 antagonist Pyr3 and TRPC1,4,5 antagonist Pico1,4,5 did not affect the standing current, whereas the TRPC blocker SKF96365 promoted rather than suppressed, Na+ influx. TM cells thus maintain the resting membrane potential, control Na+ homeostasis, and balance K+ efflux through a novel constitutive monovalent cation leak current with properties not unlike those of TRP channels. Yet to be identified at the molecular level, this novel channel sets the homeostatic steady-state and controls the magnitude of pressure-induced transmembrane signals.

Localization of Piezo 1 and Piezo 2 in Lateral Line System and Inner Ear of Zebrafish (Danio rerio).

Piezo proteins have been identified as mechanosensitive ion channels involved in mechanotransduction. Several ion channel dysfunctions may be associated with diseases (including deafness and pain); thus, studying them is critical to understand their role in mechanosensitive disorders and to establish new therapeutic strategies. The current study investigated for the first time the expression patterns of Piezo proteins in zebrafish octavolateralis mechanosensory organs. Piezo 1 and 2 were immunoreactive in the sensory epithelia of the lateral line system and the inner ear. Piezo 1 (28.7 ± 1.55 cells) and Piezo 2 (28.8 ± 3.31 cells) immunopositive neuromast cells were identified based on their ultrastructural features, and their overlapping immunoreactivity to the s100p specific marker (28.6 ± 1.62 cells), as sensory cells. These findings are in favor of Piezo proteins' potential role in sensory cell activation, while their expression on mantle cells reflects their implication in the maintenance and regeneration of the neuromast during cell turnover. In the inner ear, Piezo proteins' colocalization with BDNF introduces their potential implication in neuronal plasticity and regenerative events, typical of zebrafish mechanosensory epithelia. Assessing these proteins in zebrafish could open up new scenarios for the roles of these important ionic membrane channels, for example in treating impairments of sensory systems.

(Invited) Drug Delivery System of Photoresponsive Donor-Acceptor-Linked Molecules to Mouse Retina

Neurotransmission is mediated by electrical pulses that are generated by coordinated activation and deactivation of ion channels in the plasma membrane of neuronal cells. The ion channels control the plasma membrane potential (V m) by influx/efflux of ions. In our retina, the V m is controlled by light and photo-excitable ion channels in photoreceptor cells and voltage-dependent ion channels in neuronal ganglion cells, which transmit visual signal to the brain. In contrast, patients with visual impairment and blindness, e.g., with age-related macular degeneration, lose response to light due to irreversible elimination of photoreceptor cells. Therefore, restoring vision is potentially possible by direct photoexcitation of neuronal ganglion cells with photoresponsive V m-controlling molecules. As one of such molecules, we have focused on donor-acceptor-linked molecules (D–A molecules) capable of photoinduced intramolecular long-lived charge separation [1–4]. In this symposium, I will introduce my recent research mainly on drug delivery system of D–A molecules to the retina in mice [5].REFERENCES[1] Imahori, H. et al., J. Am. Chem. Soc. 2001, 123, 2607–2617.[2] Numata, T. et al., J. Am. Chem. Soc. 2012, 134, 6092–6095.[3] Takano, Y. et al., Chem. Sci. 2016, 7, 3331–3337.[4] Numata, T. et al., Cell. Physiol. Biochem. 2020, 54, 899–916.[5] Fukuda, R. et al., Adv. Therap. 2023, 6, 2300186.

Enhanced glycolysis causes extracellular acidification and activates acid-sensing ion channel 1a in hypoxic pulmonary hypertension.

In pulmonary hypertension (PHTN), a metabolic shift to aerobic glycolysis promotes a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs). Enhanced glycolysis induces extracellular acidosis, which can activate proton-sensing membrane receptors and ion channels. We previously reported that activation of the proton-gated cation channel acid-sensing ion channel 1a (ASIC1a) contributes to the development of hypoxic PHTN. Therefore, we hypothesize that enhanced glycolysis and subsequent acidification of the PASMC extracellular microenvironment activate ASIC1a in hypoxic PHTN. We observed decreased oxygen consumption rate and increased extracellular acidification rate in PASMCs from chronic hypoxia (CH)-induced PHTN rats, indicating a shift to aerobic glycolysis. In addition, we found that intracellular alkalization and extracellular acidification occur in PASMCs following CH and in vitro hypoxia, which were prevented by the inhibition of glycolysis with 2-deoxy-d-glucose (2-DG). Inhibiting H+ transport/secretion through carbonic anhydrases, Na+/H+ exchanger 1, or vacuolar-type H+-ATPase did not prevent this pH shift following hypoxia. Although the putative monocarboxylate transporter 1 (MCT1) and -4 (MCT4) inhibitor syrosingopine prevented the pH shift, the specific MCT1 inhibitor AZD3965 and/or the MCT4 inhibitor VB124 were without effect, suggesting that syrosingopine targets the glycolytic pathway independent of H+ export. Furthermore, 2-DG and syrosingopine prevented enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMCs from CH rats. These data suggest that multiple H+ transport mechanisms contribute to extracellular acidosis and that inhibiting glycolysis-rather than specific H+ transporters-more effectively prevents extracellular acidification and ASIC1a activation. Together, these data reveal a novel pathological relationship between glycolysis and ASIC1a activation in hypoxic PHTN.NEW & NOTEWORTHY In pulmonary hypertension, a metabolic shift to aerobic glycolysis drives a hyperproliferative, apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells. We demonstrate that this enhanced glycolysis induces extracellular acidosis and activates the proton-gated ion channel, acid-sensing ion channel 1a (ASIC1a). Although multiple H+ transport/secretion mechanisms are upregulated in PHTN and likely contribute to extracellular acidosis, inhibiting glycolysis with 2-deoxy-d-glucose or syrosingopine effectively prevents extracellular acidification and ASIC1a activation, revealing a promising therapeutic avenue.

Importance of isoleucine residue in ion channel formation ability of 11-residue peptaibols

Peptaibols are a class of short peptides, typically 7 to 20 amino acids long, characterized by noncanonical amino acid residues such as aminoisobutyric acid (Aib). Although the helix length is shorter than the membrane thickness, the 11-residue peptaibol trichorovin-XII (TV-XII) can form ion channels in membranes. Assuming that a higher proportion of isoleucine (Ile) relative to leucine (Leu) residues is crucial for maintaining the ion channel activity of TV-XII, peptide analogs of TV-XII with varying Ile content were designed, synthesized, and evaluated. The secondary structure of all derivatives under hydrophobic conditions was confirmed by CD measurement as an α-helix-like β-bend ribbon spiral structure. The most stable ion channel activity was found in compound 4a with maximum Ile. Furthermore, the C-terminal Ile analog showed greater ion channel activity compared to the Leu analog. This suggests that the choice between Leu and Ile can influence the expression of ion channel activity, which will be crucial for the de novo designed functional peptides.

Alignment of lyotropic liquid crystals using magnetic nanoparticles improves ionic transport through built-in peptide ion channels

Hypothesis: We hypothesize that simultaneous incorporation of ion channel peptides (in this case, potassium channel as a model) and hydrophobic magnetite Fe3O4 nanoparticles (hFe3O4NPs) within lipidic hexagonal mesophases, and aligning them using an external magnetic field can significantly enhance ion transport through lipid membranes.Experiments: In this study, we successfully characterized the incorporation of gramicidin membrane ion channels and hFe3O4NPs in the lipidic hexagonal structure using SAXS and cryo-TEM methods. Additionally, we thoroughly investigated the conductive characteristics of freestanding films of lipidic hexagonal mesophases, both with and without gramicidin potassium channels, utilizing a range of electrochemical techniques, including impedance spectroscopy, normal pulse voltammetry, and chronoamperometry.Findings: Our research reveals a state-of-the-art breakthrough in enhancing ion transport in lyotropic liquid crystals as matrices for integral proteins and peptides. We demonstrate the remarkable efficacy of membranes composed of hexagonal lipid mesophases embedded with K+ transporting peptides. This enhancement is achieved through doping with hFe3O4NPs and exposure to a magnetic field. We investigate the intricate interplay between the conductive properties of the lipidic hexagonal structure, hFe3O4NPs, gramicidin incorporation, and the influence of Ca2+ on K+ channels. Furthermore, our study unveils a new direction in ion channel studies and biomimetic membrane investigations, presenting a versatile model for biomimetic membranes with unprecedented ion transport capabilities under an appropriately oriented magnetic field. These findings hold promise for advancing membrane technology and various biotechnological and biomedical applications of membrane proteins.

Oxidative and Nitrous Stress Underlies Vascular Malfunction Induced by Ionizing Radiation and Diabetes.

Oxidative stress results from the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in quantities exceeding the potential activity of the body's antioxidant system and is one of the risk factors for the development of vascular dysfunction in diabetes and exposure to ionizing radiation. Being the secondary products of normal aerobic metabolism in living organisms, ROS and RNS act as signaling molecules that play an important role in the regulation of vital organism functions. Meanwhile, in high concentrations, these compounds are toxic and disrupt various metabolic pathways. The various stress factors (hyperglycemia, gamma-irradiation, etc.) trigger free oxygen and nitrogen radicals accumulation in cells that are capable to damage almost all cellular components including ion channels and transporters such as Na+/K+-ATPase, BKCa, and TRP channels. Vascular dysfunctions are governed by interaction of ROS and RNS. For example, the reaction of ROS with NO produces peroxynitrite (ONOO-), which not only oxidizes DNA, cellular proteins, and lipids, but also disrupts important signaling pathways that regulate the cation channel functions in the vascular endothelium. Further increasing in ROS levels and formation of ONOO- leads to reduced NO bioavailability and causes endothelial dysfunction. Thus, imbalance of ROS and RNS and their affect on membrane ion channels plays an important role in the pathogenesis of vascular dysfunction associated with various disorders.

SARS-CoV-2 Viroporin E Induces Ca2+ Release and Neuron Cell Death in Primary Cultures of Rat Hippocampal Cells Aged In Vitro.

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca2+ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca2+ concentration in hippocampal neurons. Ca2+ responses to the E protein are due to Ca2+ release from intracellular stores at the ER. Moreover, E protein-induced Ca2+ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca2+ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca2+, probably acting like a viroporin, thus producing Ca2+ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.

Trending toward gero‐electroceuticals that target membrane potential for reprogramming aging and lifespan

AbstractIon gradients across cell membranes generate voltage potentials that are involved in a wide range of biological processes. According to the membrane hypothesis of aging, aging is inextricably linked to a decrease in resting membrane potential (Vmem). Alterations in ion channel activity and membrane fluidity caused by aging disrupt bioelectric homeostasis, increase intracellular calcium and potassium concentrations, induce abnormal mechanistic target of rapamycin (MTOR)‐ and AMPK‐regulated metabolism and energy dissipation, and decrease proliferation and regeneration. Failure to maintain ion channel activity and membrane potential leads to cell senescence or death. There is evidence that by manipulating ion channel activities, a cryptic memory can be recalled to restore lost proliferative or regenerative abilities. Reversal or prevention of senescence, aging phenotypes, and longevity may be achieved by fine‐tuning mitochondrial membrane polarization. Therefore, there is optimism that deciphering the bioelectric codes that govern cell functions will lead to the development of new gero‐electroceuticals that restore cell function and prevent tissue loss during aging.

The TREK-1 potassium channel is a potential pharmacological target for vasorelaxation in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease in which chronic membrane potential (Em) depolarisation of the pulmonary arterial smooth muscle cells (PASMCs) causes calcium overload, a key pathological alteration. Under resting conditions, the negative Em is mainly set by two pore domain potassium (K2P) channels, of which the TASK-1 has been extensively investigated. Ion channel currents and membrane potential of primary cultured human(h) PASMCs were measured using the voltage- and current clamp methods. Intracellular [Ca2+] was monitored using fluorescent microscopy. Pulmonary BP and vascular tone measurements were also performed ex vivo using a rat PAH model. TREK-1 was the most abundantly expressed K2P in hPASMCs of healthy donors and idiopathic(I) PAH patients. Background K+-current was similar in hPASMCs for both groups and significantly enhanced by the TREK activator ML-335. In donor hPASMCs, siRNA silencing or pharmacological inhibition of TREK-1 caused depolarisation, reminiscent of the electrophysiological phenotype of idiopathic PAH. ML-335 hyperpolarised donor hPASMCs and normalised the Em of IPAH hPASMCs. A close link was found between TREK-1 activity and intracellular Ca2+-signalling using a channel activator, ML-335, and an inhibitor, spadin. In the rat, ML-335 relaxed isolated pre-constricted pulmonary arteries and significantly decreased pulmonary arterial pressure in the isolated perfused lung. These data suggest that TREK-1is a key factor in Em setting and Ca2+ homeostasis of hPASMC, and therefore, essential for maintenance of a low resting pulmonary vascular tone. Thus TREK-1 may represent a new therapeutic target for PAH.

Two co-existing and opposing mechanisms of proton transfer in one-dimensional open-end water chains.

The proton transport in one-dimensional (1D) confined water chains has been extensively studied as a model for ion channels in cell membrane and fuel cell. However, the mechanistic understanding of the proton transfer (PT) process in 1D water chains remains incomplete. In this study, we demonstrate that the two limiting structures of the hydrated excess proton, H5O2+ (Zundel) and H3O+ (linear H7O3+), undergo a change in dominance as the water chain grows, causing two co-existing and opposing PT mechanisms. Specifically, H5O2+ is stable in the middle of the chain, whereas H3O+ serves as a transition state (TS). Except for this region, H3O+ is stabilized while H5O2+ serves as a TS. The interaction analysis shows that the electrostatic interaction plays a crucial role in the difference in PT mechanisms. Our work fills a knowledge gap between the various PT mechanisms reported in bulk water and long 1D water chains, contributing to a deeper understanding of biological ion channels at the atomic level.

Spider and Wasp Acylpolyamines: Venom Components and Versatile Pharmacological Leads, Probes, and Insecticidal Agents.

Polyamines (PAs) are polycationic biogenic amines ubiquitously present in all life forms and are involved in molecular signaling and interaction, determining cell fate (e.g., cell proliferation, dif-ferentiation, and apoptosis). The intricate balance in the PAs' levels in the tissues will determine whether beneficial or detrimental effects will affect homeostasis. It's crucial to note that endoge-nous polyamines, like spermine and spermidine, play a pivotal role in our understanding of neu-rological disorders as they interact with membrane receptors and ion channels, modulating neuro-transmission. In spiders and wasps, monoamines (histamine, dopamine, serotonin, tryptamine) and polyamines (spermine, spermidine, acyl polyamines) comprise, with peptides and other sub-stances, the low molecular weight fraction of the venom. Acylpolyamines are venom components exclusively from spiders and a species of solitary wasp, which cause inhibition chiefly of iono-tropic glutamate receptors (AMPA, NMDA, and KA iGluRs) and nicotinic acetylcholine receptors (nAChRs). The first venom acylpolyamines ever discovered (argiopines, Joro and Nephila toxins, and philanthotoxins) have provided templates for the design and synthesis of numerous analogs. Thus far, analogs with high potency exert their effect at nanomolar concentrations, with high se-lectivity toward their ionotropic and ligand receptors. These potent and selective acylpolyamine analogs can serve biomedical purposes and pest control management. The structural modification of acylpolyamine with photolabile and fluorescent groups converted these venom toxins into use-ful molecular probes to discriminate iGluRs and nAchRs in cell populations. In various cases, the linear polyamines, like spermine and spermidine, constituting venom acyl polyamine backbones, have served as cargoes to deliver active molecules via a polyamine uptake system on diseased cells for targeted therapy. In this review, we examined examples of biogenic amines that play an essential role in neural homeostasis and cell signaling, contributing to human health and disease outcomes, which can be present in the venom of arachnids and hymenopterans. With an empha-sis on the spider and wasp venom acylpolyamines, we focused on the origin, structure, derivatiza-tion, and biomedical and biotechnological application of these pharmacologically attractive, chemically modular venom components.

Biophysical Mechanisms of Vaginal Smooth Muscle Contraction: The Role of the Membrane Potential and Ion Channels.

The vagina is an essential component of the female reproductive system and is responsible for providing female sexual satisfaction. Vaginal smooth muscle contraction plays a crucial role in various physiological processes, including sexual arousal, childbirth, and urinary continence. In pathophysiological conditions, such as pelvic floor disorders, aberrations in vaginal smooth muscle function can lead to urinary incontinence and pelvic organ prolapse. A set of cellular and sub-cellular physiological mechanisms regulates the contractile properties of the vaginal smooth muscle cells. Calcium influx is a crucial determinant of smooth muscle contraction, facilitated through voltage-dependent calcium channels and calcium release from intracellular stores. Comprehensive reviews on smooth muscle biophysics are relatively scarce within the scientific literature, likely due to the complexity and specialized nature of the topic. The objective of this review is to provide a comprehensive description of alterations in the cellular physiology of vaginal smooth muscle contraction. The benefit associated with this particular approach is that conducting a comprehensive examination of the cellular mechanisms underlying contractile activation will enable the creation of more targeted therapeutic agents to control vaginal contraction disorders.

Lung adenocarcinomas without driver genes converge to common adaptive strategies through diverse genetic, epigenetic, and niche construction evolutionary pathways

Somatic evolution selects cancer cell phenotypes that maximize survival and proliferation in dynamic environments. Although cancer cells are molecularly heterogeneous, we hypothesized convergent adaptive strategies to common host selection forces can be inferred from patterns of epigenetic and genetic evolutionary selection in similar tumors. We systematically investigated gene mutations and expression changes in lung adenocarcinomas with no common driver genes (n = 313). Although 13,461 genes were mutated in at least one sample, only 376 non-synonymous mutations evidenced positive evolutionary selection with conservation of 224 genes, while 1736 and 2430 genes exhibited ≥ two-fold increased and ≥ 50% decreased expression, respectively. Mutations under positive selection are more frequent in genes with significantly altered expression suggesting they often “hardwire” pre-existing epigenetically driven adaptations. Conserved genes averaged 16-fold higher expression in normal lung tissue compared to those with selected mutations demonstrating pathways necessary for both normal cell function and optimal cancer cell fitness. The convergent LUAD phenotype exhibits loss of differentiated functions and cell–cell interactions governing tissue organization. Conservation with increased expression is found in genes associated with cell cycle, DNA repair, p53 pathway, epigenetic modifiers, and glucose metabolism. No canonical driver gene pathways exhibit strong positive selection, but extensive down-regulation of membrane ion channels suggests decreased transmembrane potential may generate persistent proliferative signals. NCD LUADs perform niche construction generating a stiff, immunosuppressive microenvironment through selection of specific collagens and proteases. NCD LUADs evolve to a convergent phenotype through a network of interconnected genetic, epigenetic, and ecological pathways.

Voltage-dependent anion channels are involved in the maintenance of pig sperm quality during liquid preservation

Pigs are usually bred through artificial insemination with liquid semen preserved at 15–20 °C. While this method of preservation brings many benefits, including a greater reproductive performance compared to frozen-thawed sperm, the period of storage is a limiting factor. As the mitochondrion regulates many facets of sperm physiology, modulating its activity could have an impact on their lifespan. Aligned with this hypothesis, the present study sought to investigate whether inhibition of voltage-dependent anion channels (VDACs), which reside in the outer mitochondrial membrane and regulate the flux of ions between mitochondria and the cytosol in somatic cells, influences the resilience of pig sperm to liquid preservation at 17 °C. For this purpose, semen samples (N = 7) were treated with two different concentrations of TRO19622 (5 μM and 50 μM), an inhibitor of VDACs, and stored at 17 °C for 10 days. At days 0, 4 and 10, sperm quality and functionality parameters were evaluated by flow cytometry and computer-assisted sperm analysis (CASA). The effects of inhibiting VDACs depended on the concentration of the inhibitor. On the one hand, the greatest concentration of TRO19622 (50 μM) led to a decrease in sperm motility, viability and mitochondrial membrane potential, which could be related to the observed intracellular Ca2+ increase. In contrast, total sperm motility was higher in samples treated with 5 μM TRO19622 than in the control, suggesting that when VDACs channels are inhibited by the lowest concentration of the blocking agent the resilience of pig sperm to liquid storage increases. In conclusion, the current research indicates that mitochondrial function, as regulated by ion channels in the outer mitochondrial membrane like VDACs, is related to the sperm resilience to liquid preservation and may influence cell lifespan.

Mechanosensor channels of the lens and ciliary body: patch clamp studies

Epithelia from the lens and the ciliary body possess mechanosensors, including Piezo1 and transient receptor potential (TRP) channels, that may partake in the regulation of lens and aqueous humor volume. Although these mechanosensor channels commonly allow calcium entry, the outcomes of elevated intracellular calcium vary with the type of channel activated. For instance, separate activation of TRPV4 and TRPV1 increases Na-K-ATPase and Na/K/2Cl cotransporter activities, respectively. To further understand the role of mechanosensitivity in lens and ciliary body, we are currently using patch clamp techniques to characterize the response of primary cultured cells to chemical agonists and antagonists, as well as direct mechanical stimulation. Previously, we found that in mouse lens epithelial cells (mLEC), TRPV4 activation by agonist GSK1016790A (GSK) is followed by disappearance of a fast, transient K+-like outward current (Ipeak), decrease of a steady state outward current (Iss), fast depolarization of resting membrane potential (RMP) values from near -50 mV to near 0 mV, increase of Cx43 hemichannel-like events, and increase of overall membrane conductance ( gm) at holding potential (HP) = -50mV. More recently, we found that in mLEC, shear stress (streaming of external solution from a pipette tip over the cell membrane) reduced Ipeak but did not cause large RMP changes. Porcine non-pigmented epithelial cells (pNPE) in normal conditions display no Ipeak, small Iss, small RMP near -20 mV and spontaneous Cx43 hemichannel-like event activity. Nevertheless, GSK caused further depolarization of pNPE cells, and while no gm changes were seen when GSK was applied while holding cells at -50 mV, gm did increase when GSK exposure occurred during repeated ±80 mV pulses. Preliminary experiments show that activation of TRPM3 by its agonist pregnenolone causes a minimal hyperpolarization in mLEC. In comparison, in a line of human LEC (HLE B3) expressing TRPM3 channels, and which appear generally depolarized, pregnenolone caused a proportionally larger shift toward more negative RMP values; more intriguing, GSK has no measurable effect on the gm of HLE B3 cells. Taken together, and apart from considerations of cell- and species-specific differences, the data suggest that mechanosensor channels have different and/or opposite effects on the regulation of membrane ion channel activity, and thus on cell function. R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genomic and non-genomic action of vitamin D on ion channels – Targeting mitochondria

Recent studies revealed that mitochondria are not only a place of vitamin D3 metabolism but also direct or indirect targets of its activities. This review summarizes current knowledge on the regulation of ion channels from plasma and mitochondrial membranes by the active form of vitamin D3 (1,25(OH)2D3). 1,25(OH)2D3, is a naturally occurring hormone with pleiotropic activities; implicated in the modulation of cell differentiation, and proliferation and in the prevention of various diseases, including cancer. Many experimental data indicate that 1,25(OH)2D3 deficiency induces ionic remodeling and 1,25(OH)2D3 regulates the activity of multiple ion channels. There are two main theories on how 1,25(OH)2D3 can modify the function of ion channels. First, describes the involvement of genomic pathways of response to 1,25(OH)2D3 in the regulation of the expression of the genes encoding channels, their auxiliary subunits, or additional regulators. Interestingly, intracellular ion channels, like mitochondrial, are encoded by the same genes as plasma membrane channels. Therefore, the comprehensive genomic regulation of the channels from these two different cellular compartments we analyzed using a bioinformatic approach. The second theory explores non-genomic pathways of vitamin D3 activities. It was shown, that 1,25(OH)2D3 indirectly regulates enzymes that impact ion channels, change membrane physical properties, or directly bind to channel proteins. In this article, the involvement of genomic and non-genomic pathways regulated by 1,25(OH)2D3 in the modulation of the levels and activity of plasma membrane and mitochondrial ion channels was investigated by an extensive review of the literature and analysis of the transcriptomic data using bioinformatics.