Membrane Channels Research Articles

Cholesterol-Induced Deformation of Gramicidin A Channel Inhibits Potassium Ion Binding and Transport

Abstract Gramicidin A (gA) is a kind of antibiotic peptide produced by Bacillus brevis and it can dimerize across lipid bilayers to form a monovalent cation channel. In this work, we investigate the impact of cholesterol in the lipid bilayer on the binding of potassium ions with gA channel and the transport of the ions across the channel. The results indicate that cholesterol can significantly influence the conformational stability of the gA channel and cause the channel deformation which inhibits the potassium ion binding with the channel and transport across the channel. The work provides some molecular insights into understanding the influence of lipids on the activity of gA channel in both model membranes and plasma membranes of intact cells.

Hierarchically aligned channels in the distillation membrane enable efficient solar powered desalination

Solar-powered interfacial evaporation attracts enormous attention to solve the water crisis as a low-energy consumption, high energy conversion efficiency, and environmental-friendliness technology. Balancing water transport, water content and interfacial temperature is critical to promote evaporation performance. However, fabrication methods and investigation of dedicatedly-designed structures and channel sizes for solar-powered interfacial evaporation have been rarely reported. Herein, we propose to employ vertically aligned channels with hierarchy sizes to modulate water evaporation and energy efficiency. This unique structure is fabricated by a three-step procedure including freeze-drying, ion cross-linking, and hydrogen bond reconstruction in sequence, which achieves a membrane with vertically aligned channels ranging from 1 nm to tons of micrometers. Our solar-powered distillation membrane with finely-modulated hierarchical structure exhibits a high pure water generation rate of 2.37 kg m−2 h−1 under one sun illumination. Even when treated with seawater, it can achieve a capacity of 2.34 kg m−2 h−1 (10 wt% seawater) and stabilize a high evaporation rate for at least 50 h, which is superior to most reported solar empowered distillation membranes. Our distillation membrane features a high mechanical strength and low cost, making it an appealing candidate for the actual application in desalination.

A Bee Trp-Arg Dense Peptide with Antiproliferation Efficacy against the Prostate Cancer Cell Line DU145.

Prostate cancer accounts for 14% of male cancer-related fatalities in the UK. Given the challenges associated with hormone-based therapies in the context of androgen-independent prostate cancer, there is an imperative need for research into anticancer drugs. N0821, a peptide belonging to the Trp-Arg dense region and derived from the homologous region of various bee species, shows substantial potential for an anticancer effect. Both MTT assays and 3D spheroid assays were conducted to substantiate its antiproliferation potential and strongly indicated the antiproliferation effect of N0820 (WWWWRWWRKI) and N0821 (YWWWWRWWRKI). Notably, the mechanism underlying this effect is related to the downregulation of CCNA2 and the upregulation of CCNE1. Cell cycle arrest results from the reduction of CCNA2 in the S/G2 phase, leading to the accumulation of CCNE1. Our peptides were predicted to make an α-helix structure. This can act as an ion channel in the cell membrane. Therefore, we analyzed genes implicated in the influx of calcium ions into the mitochondria. Trp-Arg dense-region peptides are known for their antibacterial properties in targeting cell membranes, making the development of resistance less likely. Hence, further research in this area is essential and promising.

Transient Receptor Potential Ankyrin 1 (TRPA1) Modulation by 4-Hydroxynonenal (4-HNE) in Pancreatic Adenocarcinoma Cell Lines: Putative Roles for Therapies.

Transient receptor potential channels (TRP) are overexpressed in some pancreatic adenocarcinoma (PDAC) patients and cell lines, settling them as putative therapeutic targets in this disease. Reactive oxygen species (ROS), with levels increased in PDAC, modulate some members of the TRP family renamed "redox channels". Here, we investigate the direct effects of 4-hydroxinonenal (4-HNE) on TRPA1, natively expressed in PDAC cell lines and in association with cell migration and cell cycle progression. We performed microfluorimetry experiments, while the activation of resident membrane channels was investigated using confocal microscopy. We applied a prospective molecular docking of 4-HNE using Autodock and AutoDock Tools4. Also, we simulated the diffusion of 4-HNE through the membrane from the extracellular space with the Permeability of Molecules across Membranes (PerMM) web server. The analysis of cell migration was performed using the wound healing assay, and cell cycle progression was acquired using a Beckman Coulter CytoFlex flow cytometer. Our results show, for the first time in PDAC, that 4-HNE diffuses through the cell membrane and rapidly activates Ca2+ uptake in PDAC cells. This process depends on TRPA1 activation, as 4-HNE forms a covalent binding with a pocket-like region within the intracellular N-terminal of the channel, shaped by the cysteine residues 621, 641, and 665. The activation of TRPA1 by 4-HNE inhibits cell migration and induces cell cycle arrest in the G2/M phase. Our study brings new insights into the effects of 4-HNE, highlighting the activation of the TRPA1 channel, a druggable, putative target for PDAC-expressing tumors.

Do calcium channel blockers applied to cardiomyocytes cause increased channel expression resulting in reduced efficacy?

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug’s efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug’s inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug’s overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

Reliability-based thermal-fluid-structural topological optimization of three-dimensional coolant channels in proton exchange membrane fuel cells

In order to minimize the flow resistance in designing the cooling multi-channels of proton exchange membrane fuel cells (PEMFCs) under the double constraints of target thermal reliability index and target structural performance, this work proposes a three-dimensional (3D) thermal-fluid-structural reliability-based topology optimization (RBTO) method for the first time. In the RBTO for PEMFCs’ coolant channels, the position uncertainty of the multi-heat loads on the bipolar plates caused by irregular metal foam skeleton is considered, and the multi-heat load positions are treated as the interval uncertain variables within a certain range. To solve the problem of low computational efficiency caused by the coupling of topological parameter optimization and reliability constraint analysis, the RBTO is equivalently decoupled to a sequence of cycles of deterministic topology optimization (DTO) and inverse reliability assessment, and the latter is completed by performance measure approach (PMA) based on the target reliability index. Results show that RBTO introduces more flow resistance cost than DTO, and the additional flow resistance in RBTO increases with the increase of the target reliability index. The heat concentration caused by a smaller fiber diameter of foam skeletons results in more flow resistance cost of RBTO compared to DTO. When the inlet velocity of the coolant channel varies in the region of 0.05–0.3 m/s, the flow resistance of the multi-channel obtained by RBTO is 1.29–0.96 times as that of the straight multi-channel. In addition, the uneven reactant gas concentration in PEMFC gas channels leads to the uneven distribution of heat flow, and it affects the temperature distribution on bipolar plates, which in turn affects the results of RBTO. The above method and findings can be applied to design the cooling channels of PEMFCs with irregular metal foam gas channels.

Ionophore antibiotics and hop cones as regulators of digestion and metabolism in ruminants

The general characteristics of ionophore antibiotics and the mechanisms of their antimicrobial action were analyzed. Two types of ionophore antibiotics are known: those that transport ions across the membrane, and those that form a channel in the cell membrane through which ions pass. Ionophore antibiotics used in animal husbandry belong to the former group (monensin, lasalocid, salinomycin, narasin). They are synthesized by bacteria of the Streptomyces genus. Bacterial cells and rumen fluid differ in ionic composition, which is regulated by active ion transport. As a result, the cytoplasm of bacteria contains more potassium ions, while the rumen fluid, on the contrary, has more sodium ions. Ionophores transport potassium inside the cell and remove sodium outside. The bacteria try to correct this imbalance and require ATP energy to carry out this process. Eventually, energy deficit develops in the bacterial cell and it dies. Ionophore antibiotics affect only the Gram-positive bacteria in the rumen of ruminants, because they cannot penetrate through the cell wall of Gram-negative bacteria. Thus, there is a selective destruction of some types of bacteria, the most sensitive among which are the so-called hyper-ammonia-producing bacteria (HAB). Ionophore antibiotics, which change the breakdown of protein and carbohydrates, change the ratio of volatile fatty acids by increasing the proportion of propionic acid and thus inhibit methanogenesis in the rumen. Ionophore antibiotics are an important antiketotic agent because they reduce the concentration of β-hydroxybutyrate and non-esterified fatty acids in the blood of ruminants. During the transition period, cows have metabolic disorders so they are more prone to infectious diseases due to a decrease in resistance. The use of ionophore antibiotics affects the immune function and resistance to inflammatory processes, which is caused by an indirect influence associated with a lower pathological effect of ketosis and steatosis. Ionophore antibiotics reduce the negative energy balance and its pathological impact on metabolism. The influence of ionophore antibiotics on cow’s milk productivity is mostly absent or insignificant. The study presents characteristics of biologically active substances of hop cones. Hop cones contain biologically active components similar to ionophore antibiotics by action. These are prenylated flavonoids: humulone (α-acid), lupulone (β-acid) and their derivatives. These components of hop cones can be regarded as a potential substitute for ionophoric antibiotics. In particular, lupulone and some other components of hop cones inhibit the activity of Gram-positive bacteria, causing reactions similar to ionophore antibiotics. In addition, hop cones have many other biologically active compounds: phenols, essential oils, and resins, which have antimicrobial, antioxidant, sedative, phytoestrogen, insulin stimulating, immunomodulatory, and antitumor effects.

Synergistic incorporation of Ti3C2Tx with sulfonated poly(phenylene oxide) membranes: Unravelling multifaceted advancements in physicochemical, electrochemical, and antibacterial domains for enhanced electrodialytic desalination

This study delves into the solution-phase incorporation of Ti3C2Tx nanosheets into sulfonated poly(phenylene oxide) (SPPO) to prevail the conventional challenge of distant ionic channels in membranes. A series of membranes incorporating MXene (Ti3C2Tx nanosheets) at varying weight percentages (1 wt%, 2 wt%, and 5 wt%) underwent meticulous scrutiny of their physicochemical and electrochemical attributes. Among this series, MXe-02 emerged as particularly noteworthy, showing remarkable water sorption (WS= 43.8%), ion exchange capacity (IEC: 1.65 ± 0.05 meqg−1), and ionic conductivity (IC: 2.83×10−2 S cm−1), coupled with minimal dimensional change of 8.57% compared to its pristine counterpart, SPPO-B (WS=35.70%, IEC: 1.03±0.05 meqg−1, IC: 1.98×10–2 S cm−1). Advanced analytical techniques such as atomic force microscopy (AFM) and field emission-scanning electron microscopy (FE-SEM) provided insights into hydrophilic-hydrophobic phase separation, ionic domain clustering, and membrane surface attributes pivotal for enhancing electrochemical properties. Beyond optimal concentrations (>2 wt%), a significant composite-agglomeration effect hindered further advancements in physicochemical and electrochemical aspects, as verified by zeta potential measurements. The optimized MXe-02 membrane boasted a notable transport number of 0.89 with a substantial limiting current density (Ilim = 45 mA cm−2), offering a wide window for swift electrodialytic desalination. Moreover, MXe-02 membranes demonstrated an energy consumption of 0.98 kWh kg−1 and 93.52% energy efficiency, affirming their efficacy for brackish water desalination. Antibacterial properties were evaluated against Escherichia coli and Staphylococcus aureus, revealing MXe-02 and MXe-05's distinct zones of inhibition against S. aureus, highlighting their excellent antibacterial efficacy against Gram-positive bacteria. This comprehensive study shows the intricate interplay of MXene-incorporated membranes, offering valuable insights for advancing electrochemical and antibacterial performance in water desalination.

CAMP binding to closed pacemaker ion channels is cooperative

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Milestone Papers on Signal Transduction Mechanisms of Hypertension and Its Complications.

To celebrate 100 years of American Heart Association-supported cardiovascular disease research, this review article highlights milestone papers that have significantly contributed to the current understanding of the signaling mechanisms driving hypertension and associated cardiovascular disorders. This article also includes a few of the future research directions arising from these critical findings. To accomplish this important mission, 4 principal investigators gathered their efforts to cover distinct yet intricately related areas of signaling mechanisms pertaining to the pathogenesis of hypertension. The renin-angiotensin system, canonical and novel contractile and vasodilatory pathways in the resistance vasculature, vascular smooth muscle regulation by membrane channels, and noncanonical regulation of blood pressure and vascular function will be described and discussed as major subjects.

Enhanced Mono/Divalent Ion Separation via Charged Interlayer Channels in Montmorillonite-Based Membranes.

Efficient mono- and divalent ion separation is pivotal for environmental conservation and energy utilization. Two-dimensional (2D) materials featuring interlayer nanochannels exhibit unique water and ion transport properties, rendering them highly suitable for water treatment membranes. In this work, we incorporated polydopamine/polyethylenimine (PDA/PEI) copolymers into 2D montmorillonite (MMT) nanosheet interlayer channels through electrostatic interactions and bioinspired bonding. A modified laminar structure was formed on the substrate surface via a straightforward vacuum filtration. The electrodialysis experiments reveal that these membranes could achieve monovalent permselectivity of 11.06 and Na+ flux of 2.09 × 10-8 mol cm-2 s-1. The enhanced permselectivity results from the synergistic effect of electrostatic and steric hindrance effect. In addition, the interaction between the PDA/PEI copolymer and the MMT nanosheet ensures the long-term operational stability of the membranes. Theoretical simulations reveal that Na+ has a lower migration energy barrier and higher migration rate for the modified MMT-based membrane compared to Mg2+. This work presents a novel approach for the development of monovalent permselective membranes.

Phase separation and water channels in graft-type polymer electrolyte membranes for hydrogen fuel cell

Microstructural complexity in graft-type polymer electrolyte membranes upon operating conditions is still challenging for examination. Here, the phase separation and water channels of poly(styrene sulfonic acid) (PSSA)-grafted poly(ethylene-co-tetrafluoroethylene) (ETFE) polymer electrolyte membranes (ETFE-PEMs) are characterized by a newly applying method based on electron density fluctuation, F, in the small angle X-ray scattering (SAXS) profiles. The ETFE-PEMs exhibit an increase in phase separation in the ion exchange capacity of 0–0.7 mmol/g as qualitatively determined by the increase in the SAXS invariant. The increase in the SAXS invariant is attributed to the evolution of the newly separated phase between the surfaces of crystalline ETFE backbones and the side chains of PSSA grafts. This phase separation allows the formation of water channels upon low relative humidity (RH). The ionic-domain spacing is around 1.52 nm at dry condition and then increases to 1.69 nm at low RH of 10%, reflecting the ion-water aggregation to make larger aggregate size. At 30% relative humidity, the highly ordered lamellar proton-conducting nanochannels (water channels) are formed in the membrane, which correspond to the dramatic increase in proton conductivity. It is interesting to note that these conducting nanochannels remain unchanged upon 30–70% RH. Also, in this range of RH the similar signatures in the pair distribution function suggests that the molecular packing and local structures are highly similar. Above features of phase separation and water channels can elucidate important characteristics of the ETFE-PEMs, especially the high proton conductivity at low RH.

Ion Channels in Critical Membranes: Clustering, Cooperativity, and Memory Effects

Much progress has been made in elucidating the inner workings of voltage-gated ion channels, but less understood is the influence of lipid rafts on gating kinetics. Here we propose that state-dependent channel affinity for different lipid species provides a unified explanation for the experimentally observed behaviors of clustering, cooperativity, and hysteresis. We develop models of diffusing lipids and channels engaged in Ising-like interactions to investigate the collective behaviors driven by raft formation in critical membranes close to the demixing transition. The model channels demonstrate lipid-mediated long-range interactions, activation curve steepening, and long-term memory in ionic currents. These behaviors likely play a role in channel-mediated cellular signaling and suggest a universal mechanism for self-organization of biomolecular assemblies. Published by the American Physical Society 2024

Blockage of aquaporin-3 peroxiporin activity by organogold compounds affects melanoma cell adhesion, proliferation and migration.

Aquaporin-3 (AQP3) is a membrane channel with dual aquaglyceroporin/peroxiporin activity, facilitating the diffusion of water, glycerol and H2O2 across cell membranes. AQP3shows aberrant expression in melanoma and its role in cell adhesion, migration and proliferation is well described. Gold compounds were shown to modulate AQP3 activity with reduced associated toxicity, making them promising molecules for cancer therapy. In this study, we validated the phenotype resulting from AQP3-silencing of two melanoma cell lines, MNT-1 and A375, which resulted in decreased H2O2 permeability. Subsequently, the AQP3 inhibitory effect of a new series of organogold compounds derived from Auphen, a potent AQP3 inhibitor, was first evaluated in red blood cells (RBCs) that highly express AQP3, and then in HEK-293T cells with AQP3 overexpression to ascertain the compounds' specificity. The first screening in RBCs unveiled two organogold compounds as promising blockers of AQP3 permeability. Moderate reduction of glycerol permeability but drastic inhibition of H2O2 permeability was detected for some of the gold derivatives in both AQP3-overexpressing cells and human melanoma cell lines. Additionally, all compounds were effective in impairing cell adhesion, proliferation and migration, although in a cell type-dependent manner. In conclusion, our data show that AQP3 peroxiporin activity is crucial for melanoma progression and highlight organogold compounds as promising AQP3 inhibitors with implications in melanoma cell adhesion, proliferation and migration, unveiling their potential as anticancer drugs against AQP3-overexpressing tumours. KEY POINTS: AQP3 affects cellular redox balance. Gold compounds inhibit AQP3 permeability in melanoma cells. AQP3 is involved in cell adhesion, proliferation and migration of melanoma. Blockage of AQP3 peroxiporin activity impairs melanoma cell migration. Gold compounds are potential anticancer drug leads for AQP3-overexpressing cancers.

The function of mechanosensitive channels in the asymmetric lipid-protein membrane

The function of mechanosensitive channels in the asymmetric lipid-protein membrane

Boron nitride nanotube porins as a new biomimetic membrane channel for nanofluidic transport

Boron nitride nanotube porins as a new biomimetic membrane channel for nanofluidic transport

Hydrodynamic effects of the elliptical spacer filament on the flow and mass transfer in a desalination membrane channel

This study numerically investigates the impacts of an elliptical spacer on the flow and mass transfer within a desalination membrane channel. We consider the effects of the fluid Reynolds number, the elliptical spacer position yin/H, the flow attack angle θ and the aspect ratio B of spacer on the fluid flow, concentration polarization, and permeation flux. Our findings reveal that the elliptical spacers can improve both the mass transfer and permeation flux compared with the traditional circular spacers. Placing the spacer near the membrane surface effectively disrupts the concentration boundary layer, thereby amplifying the local concentration polarization surrounding the spacer. A single recirculation vortex behind the spacer induces numerous large vortices around the membrane, resulting in a significant increment in freshwater production. By changing θ and B, the system's stability can be reduced, potentially leading to a 16.0% increment in permeation flux when compared with the traditional circular spacers. Furthermore, the drag and lift coefficients acting on the elliptical spacer are greater than those of circular spacers, which is the main reason for the transition of flow condition. Our findings provide a reference for the design of the efficient structures in reverse osmosis membranes.

Na+/K+-ATPase: ion pump, signal transducer, or cytoprotective protein, and novel biological functions.

Na+/K+-ATPase is a transmembrane protein that has important roles in the maintenance of electrochemical gradients across cell membranes by transporting three Na+ out of and two K+ into cells. Additionally, Na+/K+-ATPase participates in Ca2+-signaling transduction and neurotransmitter release by coordinating the ion concentration gradient across the cell membrane. Na+/K+-ATPase works synergistically with multiple ion channels in the cell membrane to form a dynamic network of ion homeostatic regulation and affects cellular communication by regulating chemical signals and the ion balance among different types of cells. Therefore, it is not surprising that Na+/K+-ATPase dysfunction has emerged as a risk factor for a variety of neurological diseases. However, published studies have so far only elucidated the important roles of Na+/K+-ATPase dysfunction in disease development, and we are lacking detailed mechanisms to clarify how Na+/K+-ATPase affects cell function. Our recent studies revealed that membrane loss of Na+/K+-ATPase is a key mechanism in many neurological disorders, particularly stroke and Parkinson's disease. Stabilization of plasma membrane Na+/K+-ATPase with an antibody is a novel strategy to treat these diseases. For this reason, Na+/K+-ATPase acts not only as a simple ion pump but also as a sensor/regulator or cytoprotective protein, participating in signal transduction such as neuronal autophagy and apoptosis, and glial cell migration. Thus, the present review attempts to summarize the novel biological functions of Na+/K+-ATPase and Na+/K+-ATPase-related pathogenesis. The potential for novel strategies to treat Na+/K+-ATPase-related brain diseases will also be discussed.

The antiseizure medication valproate increases hemichannel activity found in brain cells, which could worsen disease outcomes.

Glial cells play relevant roles in neuroinflammation caused by epilepsy. Elevated hemichannel (HC) activity formed by connexins (Cxs) or pannexin1 (Panx1) largely explains brain dysfunctions commonly caused by neuroinflammation. Glia express HCs formed by Cxs 43, 30, or 26, while glia and neurons both express HCs formed by Panx1. Cx43 HCs allow for the influx of Ca2+, which promotes glial reactivity, enabling the release of the gliotransmitters that contribute to neuronal over-stimulation. Valproate (VPA), an antiseizure medication, has pleiotropic actions on neuronal molecular targets, and their action on glial cell HCs remains elusive. We used HeLa cells transfected with Cx43, Cx30, Cx26, or Panx1 to determine the effect of VPA on HC activity in the brain. VPA slightly increased HC activity under basal conditions, but significantly enhanced it in cells pre-exposed to conditions that promoted HC activity. Furthermore, VPA increased ATP release through Cx43 HCs. The increased HC activity caused by VPA was resistant to washout, being consistent with in silico studies, which predicted the binding site for VPA and Cx43, as well as for Panx1 HCs on the intracellular side, suggesting that VPA first enters through HCs, after which their activity increases.

Loss of activation by GABA in vertebrate delta ionotropic glutamate receptors

Ionotropic glutamate receptors (iGluRs) mediate excitatory signals between cells by binding neurotransmitters and conducting cations across the cell membrane. In the mammalian brain, most of these signals are mediated by two types of iGluRs: AMPA and NMDA (i.e. iGluRs sensitive to 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid and N-methyl-D-aspartic acid, respectively). Delta-type iGluRs of mammals also form neurotransmitter-binding channels in the cell membrane, but in contrast, their channel is not activated by neurotransmitter binding, raising biophysical questions about iGluR activation and biological questions about the role of delta iGluRs. We therefore investigated the divergence of delta iGluRs from their iGluR cousins using molecular phylogenetics, electrophysiology, and site-directed mutagenesis. We find that delta iGluRs are found in numerous bilaterian animals (e.g., worms, starfish, and vertebrates) and are closely related to AMPA receptors, both genetically and functionally. Surprisingly, we observe that many iGluRs of the delta family are activated by the classical inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Finally, we identify nine amino acid substitutions that likely gave rise to the inactivity of today's mammalian delta iGluRs, and these mutations abolish activity when engineered into active invertebrate delta iGluRs, partly by inducing receptor desensitization. These results offer biophysical insight into iGluR activity and point to a role for GABA in excitatory signaling in invertebrates.