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Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here, we present single-particle cryo-electron microscopy structures of Mus musculus LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

Many secondary active membrane transporters pump substrates against concentration gradients by coupling their uptake to symport of sodium ions. Symport requires the substrate and ions to be always transported together. Cooperative binding of the solutes is a key mechanism contributing to coupled transport in the sodium and aspartate symporter from Pyrococcus horikoshii GltPh. Here, we describe the kinetic mechanism of coupled binding for GltPh in the inward facing state. The first of the three coupled sodium ions, binds weakly and slowly, enabling the protein to accept the rest of the ions and the substrate. The last ion binds tightly, but is in rapid equilibrium with solution. Its release is required for the complex disassembly. Thus, the first ion serves to ‘open the door’ for the substrate, the last ion ‘locks the door’ once the substrate is in, and one ion contributes to both events.

Transmembrane protein 175 (TMEM175) is a K+-selective ion channel expressed in lysosomal membranes, where it establishes a membrane potential essential for lysosomal function and its dysregulation is associated with the development of Parkinson’s Disease. TMEM175 is evolutionarily distinct from all known channels, predicting novel ion-selectivity and gating mechanisms. Here we present cryo-EM structures of human TMEM175 in open and closed conformations, enabled by resolutions up to 2.6 Å. Human TMEM175 adopts a homodimeric architecture with a central ion-conduction pore lined by the side chains of the pore-lining helices. Conserved isoleucine residues in the center of the pore serve as the gate in the closed conformation. In the widened channel in the open conformation, these same residues establish a constriction essential for K+ selectivity. These studies reveal the mechanisms of permeation, selectivity and gating and lay the groundwork for understanding the role of TMEM175 in lysosomal function.

Glutamate transporters translocate their substrate-binding site across the membrane, in a cycle fueled by the energy of the sodium gradient. Now GltPh is trapped by cross-linking, with its substrate-binding site facing inward or outward, to reveal that the Na + potential is coupled to substrate binding and release, not to substrate translocation. Glutamate transporters catalyze concentrative uptake of the neurotransmitter into glial cells and neurons. Their transport cycle involves binding and release of the substrate on the extra- and intracellular sides of the plasma membranes and translocation of the substrate-binding site across the lipid bilayers. The energy of the ionic gradients, mainly sodium, fuels the cycle. Here, we used a cross-linking approach to trap a glutamate transporter homolog from Pyrococcus horikoshii in key conformational states with the substrate-binding site facing either the extracellular or the intracellular side of the membrane to study binding thermodynamics. We show that the chemical potential of sodium ions in solution is exclusively coupled to substrate binding and release, not to substrate translocation. Despite the transporter's structural symmetry, the binding mechanisms are distinct on the opposite sides of the membrane and more complex than the current models suggest.

Structures of the human lysosomal K + channel transmembrane protein 175 (TMEM175) in open and closed states revealed a novel architecture lacking the canonical K + selectivity filter motif present in previously known K + channel structures. A hydrophobic constriction composed of four isoleucine residues was resolved in the pore and proposed to serve as the gate in the closed state, and to confer ion selectivity in the open state. Here, we achieve higher-resolution structures of the open and closed states and employ molecular dynamics simulations to analyze the conducting properties of the putative open state, demonstrating that it is permeable to K + and, to a lesser degree, also Na + . Both cations must dehydrate significantly to penetrate the narrow hydrophobic constriction, but ion flow is assisted by a favorable electrostatic field generated by the protein that spans the length of the pore. The balance of these opposing energetic factors explains why permeation is feasible, and why TMEM175 is selective for K + over Na + , despite the absence of the canonical selectivity filter. Accordingly, mutagenesis experiments reveal an exquisite sensitivity of the channel to perturbations that mitigate the constriction. Together, these data reveal a novel mechanism for selective permeation of ions by TMEM175 that is unlike that of other K + channels.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) remains a major global health concern owing to its multidrug resistance and persistence despite continued antibiotic development. Eco-friendly nanomaterials such as selenium nanoparticles (SeNPs) have emerged as promising antimicrobial alternatives because of their high biocompatibility and lower toxicity compared to conventional metallic nanoparticles. In this study, we investigated the inhibitory effects and underlying mechanisms of Lactiplantibacillus plantarum (LP)–derived SeNPs (LP-SeNPs) on MRSA. Methods: SeNPs were biosynthesized using the antibacterial cell-free supernatant (CFS) of LP, which provides naturally reducing and stabilizing biomolecules. The resulting LP-SeNPs were characterized by physicochemical and structural analyses and compared to chemically synthesized SeNPs (Chem-SeNPs). Antibacterial activity was assessed through minimum inhibitory concentration (MIC) testing, time-kill kinetics, and cell viability assays. Results: LP-SeNPs, which were spherical with an average diameter of 107 nm, exhibited selective antibacterial activity against Gram-positive bacteria and showed no effect on Gram-negative strains. Notably, all six MRSA isolates demonstrated high susceptibility, with MIC values approximately 100-fold lower than that of S. aureus ATCC 25923, a non-MRSA reference strain. LP-SeNPs were also non-cytotoxic up to 20-fold the MIC (IC50 > 10 µg/mL). Mechanistic analyses indicated that disruption of the bacterial cell membrane was the primary antibacterial mechanism, supported by additional contributions from reactive oxygen species generation and protein synthesis inhibition. Conclusions: LP-SeNPs represent a sustainable, biocompatible, and potent antibacterial nanoplatform with strong selectivity for Gram-positive pathogens, particularly MRSA. These findings highlight their potential as eco-friendly and targeted therapeutic strategies for combating MRSA infections.

Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues. Molecules of glutamate can carry messages between cells in the brain, and these signals are essential for thought and memory. Glutamate molecules can also act as signals to build new connections between brain cells and to prune away unnecessary ones. However, too much glutamate outside of the cells kills the brain tissue and can lead to devastating brain diseases. In a healthy brain, special pumps called glutamate transporters move these molecules back into the brain cells, where they can be stored safely. However, when brain cells are damaged—by, for example, a stroke or an injury,—the glutamate stored inside spills out, killing the surrounding cells. This leads to a cascade of dying cells and leaking glutamate, which causes even more damage and slows the recovery. Glutamate transporters ensure that there are more glutamate molecules inside cells than outside. However, it requires energy to maintain this gradient in the concentration of glutamate molecules. The transporters get this energy by moving three sodium ions into the cell with each glutamate molecule, and moving one potassium ion out of the cell. However, it is not clear how these transporters ensure that they move the glutamate molecules and the sodium ions at the same time. Now, Verdon, Oh et al. have uncovered the 3D structure of a glutamate transporter homologue at each step of the transport process. These structures reveal that, on the outside of the cell membrane, sodium ions attach to the so-called ‘transporter domain’ and make it better able to bind glutamate. The transporter domain then carries the sodium ions and glutamate through the cell membrane and releases them into the cell. Verdon, Oh et al. suggest that a potassium ion then binds to the empty transport domain, stabilizing it into a more compact shape that easily makes the return trip to the outside of the cell. Most experiments on glutamate transporters, including the work of Verdon, Oh et al., are carried out on model proteins taken from bacteria. An important challenge for the future will be to obtain structural information on human glutamate transporters, as these could be therapeutic targets for the treatment of various neurological conditions.

Transmembrane protein 175 (TMEM175) is an evolutionarily distinct lysosomal cation channel whose mutation is associated with the development of Parkinson’s disease. Here, we present a cryoelectron microscopy structure and molecular simulations of TMEM175 bound to 4-aminopyridine (4-AP), the only known small-molecule inhibitor of TMEM175 and a broad K⁺ channel inhibitor, as well as a drug approved by the Food and Drug Administration against multiple sclerosis. The structure shows that 4-AP, whose mode of action had not been previously visualized, binds near the center of the ion conduction pathway, in the open state of the channel. Molecular dynamics simulations reveal that this binding site is near the middle of the transmembrane potential gradient, providing a rationale for the voltage-dependent dissociation of 4-AP from TMEM175. Interestingly, bound 4-AP rapidly switches between three predominant binding poses, stabilized by alternate interaction patterns dictated by the twofold symmetry of the channel. Despite this highly dynamic binding mode, bound 4-AP prevents not only ion permeation but also water flow. Together, these studies provide a framework for the rational design of novel small-molecule inhibitors of TMEM175 that might reveal the role of this channel in human lysosomal physiology both in health and disease.

Transmembrane protein 175 (TMEM175) is a K.sup.+-selective ion channel expressed in lysosomal membranes, where it establishes a membrane potential essential for lysosomal function and its dysregulation is associated with the development of Parkinson's Disease. TMEM175 is evolutionarily distinct from all known channels, predicting novel ion-selectivity and gating mechanisms. Here we present cryo-EM structures of human TMEM175 in open and closed conformations, enabled by resolutions up to 2.6 Å. Human TMEM175 adopts a homodimeric architecture with a central ion-conduction pore lined by the side chains of the pore-lining helices. Conserved isoleucine residues in the center of the pore serve as the gate in the closed conformation. In the widened channel in the open conformation, these same residues establish a constriction essential for K.sup.+ selectivity. These studies reveal the mechanisms of permeation, selectivity and gating and lay the groundwork for understanding the role of TMEM175 in lysosomal function.