Membrane Channels Research Articles

Activation of connexin hemichannels enhances mechanosensitivity and anabolism in disused and aged bone.

Mechanical loading, essential for bone health, promotes bone formation and remodeling. However, the positive response diminishes in cases of disuse and aging, leading to bone loss and an increased fracture risk. This study demonstrates that activating hemichannels (HCs) using a connexin 43 (Cx43) antibody, Cx43(M2), in bone osteocytes revitalizes aging and disused bones. Using a hindlimb suspension (HLS) disuse model and a tibial mechanical loading model, we found that Cx43(M2) inhibited bone loss and osteocyte apoptosis induced by unloading in 16-week-old adult mice. Additionally, it enhanced bone mass in response to tibial loading in 22-month-old aged mice. The HC opening released bone anabolic factor prostaglandin E2 (PGE2) and suppressed catabolic factor sclerostin (SOST). This suppressed the increase of cortical bone formation and reduction of bone resorption during unloading and promoted trabecular and cortical bone formation during loading. Cx43(M2)-induced HC opening, coupled with PGE2 release, effectively rescued unloading-induced bone loss and restored the diminished anabolic response of aged bones to mechanical loading. Activating HCs with the Cx43 antibody holds promise as a de novo therapeutic approach, as it can overcome the limitations of existing treatment regimens for treating bone loss and osteoporosis associated with aging and disuse.

A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model

Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability (“leaky”) or excessive activity (“hyperactive”).We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca2+ uptake in response to elevated extracellular Ca2+ concentrations ([Ca2+]ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca2+ uptake, achieving a 50% inhibitory concentration (EC50) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations.In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody’s applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs.

Facile fabrication of 6FDA-DAM/PGMA blend membranes for advanced gas separation

Polymer blend membranes are an efficient and cost-effective route to improve the gas separation performance. The poor solubility and compatibility of the polymer are the main challenge to overcome in blend membranes procedures. Here, the incorporation of electron beam (EB) irradiation-induced polymer to membranes were reported to successfully avoid the poor solubility and compatibility between polymer. Specifically, small molecule glycidyl methacrylate (GMA) is dispersed into 6FDA-DAM membrane, 6FDA-DAM/PGMA blend membranes are obtained via EB-irradiation-polymerized GMA to form PGMA and deposit in DAM membrane in-situ. The loads of PGMA in 6FDA-DAM membrane (7.3 %-30.5 %) were controlled by EB-irradiation dosagess (from 60 kGy to 120 kGy). The resulted 6FDA-DAM/PGMA-x blend membranes showed a strong molecular sieve, such as the CO2/CH4 and H2/CH4 selectivity of 6FDA-DAM/PGMA-21.7 % membranes are improved by 352 % and 662 % compared with 6FDA-DAM, which exceed 2008 and 2015 upper bound. The enhanced performance results from incorporation of PGMA to form sub-nanometre-sized channel in 6FDA-DAM membrane. This work provides a facile and universal strategy to induce small molecule polymerization and in-situ incorporate to the membrane via EB-irradiation technology for gas separation.

Threading MOF membranes with polymer chains for superior benzene/cyclohexane separation

High performance membranes for benzene/cyclohexane separation are crucial. Metal–organic frameworks (MOFs), given by their high structural designability, are expected to provide satisfying membrane performance for this separation. In this study, polymer chains were threaded into the pores of MOF UiO-66, to reconstruct the structures of the membrane channels. The permeance of benzene and selectivity of benzene/cyclohexane were boosted simultaneously, compared with the bare UiO-66 membranes. The performance enhancement was rationalized since the solubility of benzene was improved meanwhile the diffusivity of cyclohexane dropped by virtue of the new adsorption sites of benzene and narrower membrane channels created by threading polymers. The as-synthesized polyvinyl alcohol (PVA)-threaded UiO-66 membranes exhibited a benzene permeance around 110 GPU and a benzene/cyclohexane selectivity around 30.

Anti‑angiogenic effect of Bryopsis plumosa‑derived peptide via aquaporin 3 in non‑small cell lung cancer.

Developing novel anti‑angiogenic agents with minimal toxicity is notably challenging for cancer therapeutics. The discovery and development of peptides, whether derived from natural sources or synthesized, has potential for developing anti‑angiogenic agents characterized by their ability to penetrate cancer cells, high specificity and low toxicity. The present study identified a Bryopsis plumose‑derived anticancer and anti‑angiogenesis marine‑derived peptide 06 (MP06). A 22‑amino acid peptide was synthesized and conjugated with fluorescein isothiocyanate (FITC‑MP06) for intracellular localization in H1299 non‑small cell lung cancer cells. Regulatory effects of this peptide on the viability, migration and self‑renewal of lung cancer cells was assessed. Furthermore, anti‑angiogenic effect of MP06 was investigated by monitoring vascular tube formation in human umbilical vein endothelial cells and a zebrafish model. Aquaporin (AQP)3, a membrane channel in various tissues, is involved in regulating stemness, epithelial‑mesenchymal transition (EMT) and angiogenesis. MP06 downregulated AQP3 expression. Consistently, AQP3 knockdown by RNA silencing downregulated its gene expression, leading to a decrease in stemness, EMT and angiogenesis properties in H1299 cells. MP06 could thus serve as a novel therapeutic target with anticancer and angiogenesis properties for non‑small cell lung cancer.

AChiral COFs Membrane for Enantioselective Amino Acid Separation.

Incorporating chiral molecules in the covalent organic frameworks (COFs) with uniformly ordered pores results in chiral COFs, which have been highly promising candidates for enantioseparation. Herein, a homochiral COF nanochannel membrane is reported by introducing chiral centers (L-phenylalanine methyl ester) into one of the organic ligands for the enantioseparation of chiral amino acids. The separation results show that the D-isomer is preferentially transported through the porous membrane channel. This composite membrane exhibits excellent selectivity for racemic phenylalanine with the highest enantiomeric excess value of up to 99.4%. The adsorption and molecular modeling studies substantiate the experiment results by showing higher bonding affinity towards D-isomer over L-isomer. The excellent enantioselective gating performance unveils the porous COF skeleton with chiral selectors and the size-matching synergy for stereoselective interactions with chiral isomers.

Cubic-like Features of I–V Relations via Classical Poisson–Nernst–Planck Systems Under Relaxed Electroneutrality Boundary Conditions

We focus on higher-order matched asymptotic expansions of a one-dimensional classical Poisson–Nernst–Planck system for ionic flow through membrane channels with two oppositely charged ion species under relaxed electroneutrality boundary conditions. Of particular interest are the current–voltage (I–V) relations, which are used to characterize the two most relevant biological properties of ion channels—permeation and selectivity—experimentally. Our result shows that, up to the second order in ε=λ/r, where λ is the Debye length and r is the characteristic radius of the channel, the cubic I–V relation has either three distinct real roots or a unique real root with a multiplicity of three, which sensitively depends on the boundary layers because of the relaxation of the electroneutrality boundary conditions. This indicates more rich dynamics of ionic flows under our more realistic setups and provides a better understanding of the mechanism of ionic flows through membrane channels.

Aquaporins in Biliary Function: Pathophysiological Implications and Therapeutic Targeting.

Aquaporins (AQPs) are transmembrane proteins permeable to water and a series of small solutes. AQPs play a key role in pathways of hepatobiliary secretion at the level of the liver, bile ducts, and gallbladder. AQP8 and -9 are pivotal in facilitating the osmotic water movement of hepatic bile, which is composed of 95% water. In the biliary tract, AQP1 and -4 are involved in the rearrangement of bile composition by mechanisms of reabsorption/secretion of water. In the gallbladder, AQP1 and -8 are also involved in trans-epithelial bidirectional water flow with the ultimate goal of bile concentration. Pathophysiologically, AQPs have been indicated as players in several hepatobiliary disorders, including cholestatic diseases and cholesterol cholelithiasis. Research on AQP function and the modulation of AQP expression is in progress, with the identification of potent and homolog-specific compounds modulating the expression or inhibiting these membrane channels with promising pharmacological developments. This review summarizes the contribution of AQPs in physiological and pathophysiological stages related to hepatobiliary function.

A two-faced membrane channel.

Contrasting surface properties activate a feedback loop system for a complete oil-and-water separation.

Action potential threshold variability for different electrostimulation models and its potential impact on occupational exposure limit values.

Occupational exposure limit values (ELVs) for body internal electric fields can be derived from thresholds for action potential generation. These thresholds can be calculated with electrostimulation models. The spatially extended nonlinear node model (SENN) is often used to determine such thresholds. Important parameters of these models are the membrane channel dynamics describing the ionic transmembrane currents as well as the temperature at which the models operate. This work compares action potential thresholds for five different membrane channel dynamics used with the SENN model. Furthermore, two more detailed double-cable models by Gaines et al. (MRG-Sensory and MRG-Motor) are also considered in this work. Thresholds calculated with the SENN model and the MRG models are compared for frequencies between 1 Hz and 100 kHz and temperatures at 22°C and 37°C. Results show that MRG thresholds are lower than SENN thresholds. Deriving alternative ELVs from these thresholds shows that the alternative ELVs can change significantly with different ion channel dynamics (up to a factor of 22). Using the double cable model could lead to approximately ten times lower alternative exposure limit values. On the contrary, using the SENN model with different membrane channel dynamics could also lead to higher alternative exposure limit values. Therefore, future exposure guidelines should take the influence of different electrostimulation models into account when deriving ELVs.

Paralichthys olivaceus GSDME-mediated pyroptosis is regulated by multiple caspases in different manners

Pyroptosis is a type of programmed cell death mediated by gasdermin (GSDM). GSDM is activated by caspase (CASP), which cleaves GSDM to release the N-terminal (NT) fragment that forms channels in the plasma membrane and leads to cell death. To date, research on pyroptosis in teleost is limited. In this study, we examined the activation and regulation mechanism of pyroptosis in flounder Paralichthys olivaceus. P. olivaceus gasdermin E (PoGSDME) was found to be cleaved by six P. olivaceus caspases (PoCASP1/3a/3b/7/8a/8b). PoCASP1/3a/3b/7 cleaved primarily at 245FEAD248, which generated an NT fragment (NT248) that induced robust pyroptosis. PoCASP8a/8b cleaved both the full length PoGSDME and NT248 at 202IEKD205, thus destroying the biological activity of PoGSDME and NT248. Nine residues crucial for PoGSDME function were identified, of which, F2, L19, and G85 were essential to plasma membrane translocation. During bacterial infection, PoGSDME and PoCASP1 expressions were significantly upregulated in flounder tissues, and PoGSDME, as well as PoCASP1, activation occurred in flounder cells accompanied with the processing cleavage of IL-1β and IL-18. Together these results revealed both the activation and the inhibition mechanisms of GSDME-mediated pyroptosis in flounder, and added new insights into the regulation of pyroptosis in fish.

Distinct Strategies Regulate Correlated Ion Channel mRNAs and Ionic Currents in Continually versus Episodically Active Neurons.

Relationships among membrane currents allow central pattern generator (CPG) neurons to reliably drive motor programs. We hypothesize that continually active CPG neurons utilize activity-dependent feedback to correlate expression of ion channel genes to balance essential membrane currents. However, episodically activated neurons experience absences of activity-dependent feedback and, thus, presumably employ other strategies to coregulate the balance of ionic currents necessary to generate appropriate output after periods of quiescence. To investigate this, we compared continually active pyloric dilator (PD) neurons with episodically active lateral gastric (LG) CPG neurons of the stomatogastric ganglion (STG) in male Cancer borealis crabs. After experimentally activating LG for 8 h, we measured three potassium currents and abundances of their corresponding channel mRNAs. We found that ionic current relationships were correlated in LG's silent state, but ion channel mRNA relationships were correlated in the active state. In continuously active PD neurons, ion channel mRNAs and ionic currents are simultaneously correlated. Therefore, two distinct relationships exist between channel mRNA abundance and the ionic current encoded in these cells: in PD, a direct correlation exists between Shal channel mRNA levels and the A-type potassium current it carries. Conversely, such channel mRNA-current relationships are not detected and appear to be temporally uncoupled in LG neurons. Our results suggest that ongoing feedback maintains membrane current and channel mRNA relationships in continually active PD neurons, while in LG neurons, episodic activity serves to establish channel mRNA relationships necessary to produce the ionic current profile necessary for the next bout of activity.

Monocyte-derived extracellular vesicles, stimulated by Trypanosoma cruzi, enhance cellular invasion invitro via activated TGF-β1.

During cell invasion, large Extracellular Vesicle (lEV) release from host cells was dose-dependently triggered by Trypanosoma cruzi metacyclic trypomastigotes (Mtr). This lEV release was inhibited when IP3-mediated Ca2+ exit from the ER and further Ca2+ entry from plasma membrane channels was blocked, but whilst any store-independent Ca2+ entry (SICE) could continue unabated. That lEV release was equally inhibited if all entry from external sources was blocked by chelation of external Ca2+ points to the major contributor to Mtr-triggered host cell lEV release being IP3/store-mediated Ca2+ release, SICE playing a minor role. Host cell lEVs were released through Mtr interaction with host cell lipid raft domains, integrins, and mechanosensitive ion channels, whereupon [Ca2+]cyt increased (50 to 750nM) within 15 s. lEV release and cell entry of T. cruzi, which increased up to 30 and 60 mpi, respectively, as well as raised actin depolymerization at 60 mpi, were all reduced by TRPC inhibitor, GsMTx-4. Vesicle release and infection was also reduced with RGD peptide, methyl-β-cyclodextrin, knockdown of calpain and with the calpain inhibitor, calpeptin. Restoration of lEV levels, whether with lEVs from infected or uninfected epithelial cells, did not restore invasion, but supplementation with lEVs from infected monocytes, did. We provide evidence of THP-1 monocyte-derived lEV interaction with Mtr (lipid mixing by R18-dequenching; flow cytometry showing transfer to Mtr of R18 from R18-lEVs and of LAP(TGF-β1). Active, mature TGF-β1 (at 175 pg/×105 in THP-1 lEVs) was detected in concentrated lEV-/cell-free supernatant by western blotting, only after THP-1 lEVs had interacted with Mtr. The TGF-β1 receptor (TβRI) inhibitor, SB-431542, reduced the enhanced cellular invasion due to monocyte-lEVs.

Electrochemical identification and quantification of through-plane proton channels in graphene oxide membranes.

Stacked graphene oxide (GO) proton membranes are promising candidates for use in energy devices due to their proton conductivity. Identification of through-plane channels in these membranes is critical but challenging due to their anisotropic nature. Here, we present an electrochemical reduction method for identifying and quantifying through-plane proton channels in GO membranes. The simplicity lies in the operando optical observation of the change in contrast as GO is electrochemically reduced. Here, we find three proton-dominated three-phase interfaces, which are critical for the reduction reactions of GO membranes. Based on these findings, a method is proposed to identify and quantify through-plane channels in stacked GO proton membranes using a simple three-electrode device in combination with real-time imaging of the membrane surface.

PilY1 regulates the dynamic architecture of the type IV pilus machine in Pseudomonas aeruginosa

Type IV pili (T4P) produced by the pathogen Pseudomonas aeruginosa play a pivotal role in adhesion, surface motility, biofilm formation, and infection in humans. Despite the significance of T4P as a potential therapeutic target, key details of their dynamic assembly and underlying molecular mechanisms of pilus extension and retraction remain elusive, primarily due to challenges in isolating intact T4P machines from the bacterial cell envelope. Here, we combine cryo-electron tomography with subtomogram averaging and integrative modelling to resolve in-situ architectural details of the dynamic T4P machine in P. aeruginosa cells. The T4P machine forms 7-fold symmetric cage-like structures anchored in the cell envelope, providing a molecular framework for the rapid exchange of major pilin subunits during pilus extension and retraction. Our data suggest that the T4P adhesin PilY1 forms a champagne-cork-shaped structure, effectively blocking the secretin channel in the outer membrane whereas the minor-pilin complex in the periplasm appears to contact PilY1 via the central pore of the secretin gate. These findings point to a hypothetical model where the interplay between the secretin protein PilQ and the PilY1-minor-pilin priming complex is important for optimizing conformations of the T4P machine in P. aeruginosa, suggesting a gate-keeping mechanism that regulates pilus dynamics.

TRPV4-A Multifunctional Cellular Sensor Protein with Therapeutic Potential.

Transient receptor potential vanilloid (TRPV) channel proteins belong to the superfamily of TRP proteins that form cationic channels in the animal cell membranes. These proteins have various subtype-specific functions, serving, for example, as sensors for pain, pressure, pH, and mechanical extracellular stimuli. The sensing of extracellular cues by TRPV4 triggers Ca2+-influx through the channel, subsequently coordinating numerous intracellular signaling cascades in a spatio-temporal manner. As TRPV channels play such a wide role in various cellular and physiological functions, loss or impaired TRPV protein activity naturally contributes to many pathophysiological processes. This review concentrates on the known functions of TRPV4 sensor proteins and their potential as a therapeutic target.

Effects of droplet dynamical behavior in flow channel of proton exchange membrane fuel cell under vibration conditions on cell performance

Proton exchange membrane fuel cells are subjected to severe vibration in aerospace applications. Understanding the droplet transport characteristics in the flow channelunder vibration conditions is essential for improving cell performance. However, the underlying patterns and mechanisms of the vibration effects on cell performance and its internal water transport remain unclear, and there is a lack of improvement methods for drainage under vibration conditions. This study experimentally investigates the effect of vibration on cell performance. Experimental results indicate that vibration predominantly affects cell performance through its modulation of water transport. Therefore, the droplet dynamic behavior in the flow channel is further studied using the volume of fluid method. The results indicate that vibrations can enhance the average performance of the fuel cells, with the largest increase of 8.30 %. This is attributed to vibrations promoting the detachment of droplets from the gas diffusion layer surface and reducing the water coverage ratio on said surface. Under conditions of low gas velocity, high surface hydrophobicity, and large droplet size, droplets exhibited oscillatory and jumping behavior in the flow channel due to vibration, resulting in the largest 53.5 % increase in peak water coverage ratio on the gas diffusion layer surface compared to no-vibration conditions. Furthermore, enhancing the hydrophobicity of the gas diffusion layer and the hydrophilicity of the channel walls promotes droplet detachment and discharge compared to no-vibration, as well as contributing to the improvement of vibration effects on the drainage process.

Vitamin D in cardiac fibroblasts: new evidence for a TRP-mediated non-genomic pathway

Abstract Background/Introduction Vitamin D’s (VD) role transcends phosphocalcic metabolism to affect different systems, notably the cardiovascular system in health and diseases. Beyond its genomic effect, VD act through non-genomic calcium (Ca2+) signaling pathways, hinting at a broader spectrum of physiological impacts. Despite the established interactions with cardiomyocytes via the Vitamin D Receptor (VDR), little is known about VD and cardiac fibroblast, a pivotal cell player in cardiac function and disease. Purpose This research aims to reveal Vitamin D's role in modulating cardiac fibroblast function through Transient Receptor Potential (TRP) channels. Methods Utilizing primary cardiac fibroblasts from human and rats, our investigation focused on intracellular calcium signaling. Through Ca2+ imaging techniques combined to synthetic fluorescent VD analogue, we explored Vitamin D's behavior within the cell. The study further employed biochemical assays to elucidate the interaction between VDR on the cell membrane and TRP channels signaling, providing a detailed map of the cellular choreography orchestrated by VD within cardiac fibroblasts. In addition, in vitro and ex vivo heart perfusion experiments were incorporated to assess cardiac cGMP production in response to VD. Results Our findings reveal a unique pathway wherein VD induces Ca2+ oscillations in human and rat cardiac fibroblasts specifically through the TRPC3 channel, sidelining other calcium sources. This process is regulated by the interaction between TRPC3 and the membrane-bound VDR, harmonizing with phospholipase C signaling to a crescendo of cGMP production in cardiac fibroblasts, and across the entire heart. This was associated with less pro-fibrotic potential of cardiac fibroblasts. Conclusion This study provides the first evidence of a TRP-mediated, VD non-genomic pathway within human and rat cardiac fibroblasts. Our findings herald a promising horizon for investigating vitamin D’s antifibrotic potential in heart disease.

Constructing highly durable proton transport channels in polybenzimidazole membranes using highly stable porous carbonized metal organic frameworks

The use of porous materials to construct proton transport channels in high-temperature proton exchange membranes (HT-PEMs) is an effective strategy for improving cell performance. However, most reported metal organic frameworks (MOFs) struggle to maintain stability in highly acidic or alkaline environments, which hinders their application. In this work, highly stable carbonized MOFs based on MIL-101(Cr) are prepared and used as porous materials to construct proton transport channels in HT-PEMs. The porous carbon derived from MOFs exhibits new structures and properties owing to the periodic porous and hybrid structure used to prepare porous carbon with uniform pores. High specific surface area and highly stable proton transport channels are constructed in a phosphoric acid (PA)-doped polybenzimidazole system. The tensile properties and anti-oxidization capacity of prepared membranes are improved as the doping content of carbonized MOFs increased. Meanwhile, the conductivities of prepared membranes increase with increasing amounts of the carbonized MOFs at a similar acid doping level. The membrane noted CM15@OPBI achieves a conductivity of 0.059 S cm−1 and a power density of 648 mW cm−2 at 0 % RH, 160 °C (H2/O2). Moreover, the membrane stably withstands a constant current discharge of 200 mA cm−2 for 200 h under those conditions.

ATP-release pannexin channels are gated by lysophospholipids.

In addition to its role as cellular energy currency, adenosine triphosphate (ATP) serves as an extracellular messenger that mediates diverse cell-to-cell communication. Compelling evidence supports that ATP is released from cells through pannexins, a family of membrane proteins that form heptameric large-pore channels. However, the activation mechanisms that trigger ATP release by pannexins remain poorly understood. Here, we discover lysophospholipids as endogenous pannexin activators, using activity-guided fractionation of mouse tissue extracts combined with untargeted metabolomics and electrophysiology. We show that lysophospholipids directly and reversibly activate pannexins in the absence of other proteins. Secretomics experiments reveal that lysophospholipid-activated pannexin 1 leads to the release of not only ATP but also other signaling metabolites, such as 5'-methylthioadenosine, which is important for immunomodulation. We also demonstrate that lysophospholipids activate endogenous pannexin 1 in human monocytes, leading to the release of IL-1β through inflammasome activation. Our results provide a connection between lipid metabolism and purinergic signaling, both of which play major roles in immune responses.