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Plant vacuolar-type Na+, K+/H+ antiporters (NHXs) play important roles in pH and K+ homeostasis and osmotic balance under normal physiological conditions. Under salt stress, vacuolar-type NHX enhances salt tolerance by compartmentalizing Na+ into vacuoles. However, the ion transport mechanism of vacuolar-type NHX remains poorly understood due to the absence of resolved protein crystal structures. To investigate the ion transport mechanism for vacuolar-type NHX, the three-dimensional structure of rice vacuolar-type NHX1 (OsNHX1) was established through homology modeling and AlphaFold3.0. The OsNHX1 model contains thirteen transmembrane segments according to hydrophobic characteristics and empirical and phylogenetic data. Furthermore, this study validated the OsNHX1 model via functional experiments, revealing a set of key charged amino acids essential for its activity. Mapping these amino acids onto the OsNHX1 model revealed that its pore domain exhibits a transmembrane charge-compensated pattern similar to that of NHE1 while also displaying a distinct charge distribution on either side of the pore domain. Comparative analysis of the key amino acid sites responsible for ion transport in the crystal structure of OsSOS1 and NHE1 revealed that OsNHX1 employs a unique ion transport mechanism. This study will enhance our understanding of the function and catalytic mechanism of OsNHX1 and other plant vacuolar-type NHXs.

Mammalian Na+/H+ exchanger type I isoform (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH (pHi) by removing one intracellular proton in exchange for one extracellular sodium ion. Abnormal activity of the protein occurs in cardiovascular disease and breast cancer. The purpose of this study is to examine the role of negatively charged amino acids of extracellular loop 3 (EL3) in the activity of the NHE protein. We mutated glutamic acid 217 and aspartic acid 226 to alanine, and to glutamine and asparagine, respectively. We examined effects on expression levels, cell surface targeting and activity of NHE1, and also characterized affinity for extracellular sodium and lithium ions. Individual mutation of these amino acids had little effect on protein function. However, mutation of both these amino acids together impaired transport, decreasing the Vmax for both Na+ and Li+ ions. We suggested that amino acids E217 and D226 form part of a negatively charged coordination sphere, which facilitates cation transport in the NHE1 protein.

Plants remodel their cells through the dynamic endomembrane system. Intracellular pH is important for membrane trafficking, but the determinants of pH homeostasis are poorly defined in plants. Electrogenic proton (H+) pumps depend on counter-ion fluxes to establish transmembrane pH gradients at the plasma membrane and endomembranes. Vacuolar-type H+-ATPase-mediated acidification of the trans-Golgi network is crucial for secretion and membrane recycling. Pump and counter-ion fluxes are unlikely to fine-tune pH; rather, alkali cation/H+ antiporters, which can alter pH and/or cation homeostasis locally and transiently, are prime candidates. Plants have a large family of predicted cation/H+ exchangers (CHX) of obscure function, in addition to the well-studied K+(Na+)/H+ exchangers (NHX). Here, we review the regulation of cytosolic and vacuolar pH, highlighting the similarities and distinctions of NHX and CHX members. In planta, alkalinization of the trans-Golgi network or vacuole by NHXs promotes membrane trafficking, endocytosis, cell expansion, and growth. CHXs localize to endomembranes and/or the plasma membrane and contribute to male fertility, pollen tube guidance, pollen wall construction, stomatal opening, and, in soybean (Glycine max), tolerance to salt stress. Three-dimensional structural models and mutagenesis of Arabidopsis (Arabidopsis thaliana) genes have allowed us to infer that AtCHX17 and AtNHX1 share a global architecture and a translocation core like bacterial Na+/H+ antiporters. Yet, the presence of distinct residues suggests that some CHXs differ from NHXs in pH sensing and electrogenicity. How H+ pumps, counter-ion fluxes, and cation/H+ antiporters are linked with signaling and membrane trafficking to remodel membranes and cell walls awaits further investigation.

The SLC9 gene family encodes Na + /H + exchangers (NHEs). These transmembrane proteins transport ions across lipid bilayers in a diverse array of species from prokaryotes to eukaryotes, including plants, fungi, and animals. They utilize the electrochemical gradient of one ion to transport another ion against its electrochemical gradient. Currently, 13 evolutionarily conserved NHE isoforms are known in mammals [ 22 , 46 , 128 ]. The SLC9 gene family (solute carrier classification of transporters: www.bioparadigms.org ) is divided into three subgroups [ 46 ]. The SLC9A subgroup encompasses plasmalemmal isoforms NHE1-5 (SLC9A1-5) and the predominantly intracellular isoforms NHE6-9 (SLC9A6-9). The SLC9B subgroup consists of two recently cloned isoforms, NHA1 and NHA2 (SLC9B1 and SLC9B2, respectively). The SLC9C subgroup consist of a sperm specific plasmalemmal NHE (SLC9C1) and a putative NHE, SLC9C2, for which there is currently no functional data [ 46 ]. NHEs participate in the regulation of cytosolic and organellar pH as well as cell volume. In the intestine and kidney, NHEs are critical for transepithelial movement of Na + and HCO 3 − and thus for whole body volume and acid–base homeostasis [ 46 ]. Mutations in the NHE6 or NHE9 genes cause neurological disease in humans and are currently the only NHEs directly linked to human disease. However, it is becoming increasingly apparent that members of this gene family contribute to the pathophysiology of multiple human diseases.

Membrane lipids such as cardiolipin act as molecular glue to preserve the oligomeric states of membrane proteins with low oligomeric stability. Membrane-protein stabilization by lipid binding It is well established that lipid binding leads to the oligomerization of membrane proteins, and hence the activation of many signalling pathways, but it is not clear how the lipid bilayer affects the structure and function of membrane–protein complexes. Carol Robinson and colleagues have developed a mass-spectroscopy-based technique that allows the observation of oligomeric membrane–protein complexes with sufficient resolution to characterize their bound lipids. Using this technique, they evaluate the strength of oligomer formation for some 125 α-helical membrane proteins, including G-protein-coupled receptors. They find that lipid binding modulates protein interfaces, perturbing their monomer–oligomer equilibria, and show that manipulation of lipid binding can modify oligomer stability. And they use modelling to investigate possible binding sites for lipid molecules at the interfaces involved in oligomerization. These findings could aid the optimization of membrane–protein complexes for structural analysis. Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways 1 but is often difficult to define 2 or predict 3 . Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT 4 , one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET 5 , another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na + /H + antiporter NhaA from E. coli , which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

Key Points Bacteria that grow optimally in a pH range of near neutral (neutralophiles) require robust mechanisms for cytoplasmic pH homeostasis in order to survive, and in some cases grow, during exposure to acidic or alkaline conditions that are well outside the pH range tolerated for cytoplasmic pH. Extremely acidophilic bacteria maintain a cytoplasmic pH of ∼6.0 while growing at pH 1.0–3.0 in settings such as mining and geothermal areas or acidic soils, and extremely alkaliphilic bacteria maintain a cytoplasmic pH that is as much as 2.3 units below an external pH range of 9.5–11.0 in settings such as alkaline soda lakes, indigo dye plants and sewage plants. Active mechanisms of pH homeostasis under acid challenge conditions include increased expression and activity of proteins or pathways that result in outward proton pumping or the consumption of cytoplasmic protons. Under alkali challenge conditions, mechanisms of pH homeostasis include active proton accumulation or generation in the cytoplasm. Deployment of these strategies and passive adjuncts to the active strategies, such as alterations in membrane permeability to protons, require major transcriptome changes that are mediated by an intricate network of pH-sensing and signalling capabilities. The Na + /H + antiporter of Escherichia coli , NhaA, is required for alkaline pH homeostasis in the presence of Na + ; in addition to its catalytic capacity to support cytoplasmic proton accumulation at high pH, the antiporter protein possesses a pH sensor domain that results in an increase in antiport by three orders of magnitude as the pH is raised from 6.5 to 8.5. Structural studies of three-dimensional crystals of purified NhaA, combined with computational and experimental analyses, have revealed structural and mechanistic features that account for its physiological efficacy. Periplasmic pH homeostasis is a unique strategy among neutralophiles. It enables Helicobacter pylori to colonize the highly acidic surface of the stomach using urease, an acid-gated urea channel (UreI) and cytoplasmic and periplasmic carbonic anhydrases to maintain a periplasmic pH of ∼6.1. The pH gating of UreI involves hydrogen bonding of periplasmic histidines with periplasmic carboxylates. A pair of two-component pH-signalling systems play critical parts in urease trafficking to the inner membrane, where, together with UreI, the enzyme facilitates urea hydrolysis and direct export of the products (CO 2 , NH 3 and NH 4 + ) to the periplasm. Acidophiles and alkaliphiles that grow optimally at extreme pH values typically have adaptations to key proton-translocating complexes (for example, respiratory and ATP synthase complexes) and to their cell surface layers, as reflected by the high and low average isoelectric points, respectively, of their surface-exposed proteins relative to those of the surface-exposed proteins of neutralophiles. These constitutive adaptations promote optimal function at extreme pH, but reduce the growth capacity at near-neutral pH, as shown for the adaptations of the proton-translocating ATP synthase and highly expressed S-layer protein of alkaliphilic Bacillus pseudofirmus OF4. Much has been learned about individual strategies for bacterial pH homeostasis and the molecules involved, but bacterial pH homeostasis is a cell-wide physiological process that deploys and integrates these strategies differently depending on other environmental factors, such as oxygen availability and salinity. The development of systems-level models will depend on further efforts to gather broad-based quantitative 'omics' information as a function of pH under different conditions, and also on more detailed molecular information about the stoichiometric, kinetic and mechanistic properties of key transporters and enzymes. In this Review, Krulwich, Sachs and Padan describe how the evolution of diverse mechanisms for pH sensing and homeostasis has enabled bacteria to survive sudden changes in external pH and to grow in environments with external pH values that would otherwise be toxic. Diverse mechanisms for pH sensing and cytoplasmic pH homeostasis enable most bacteria to tolerate or grow at external pH values that are outside the cytoplasmic pH range they must maintain for growth. The most extreme cases are exemplified by the extremophiles that inhabit environments with a pH of below 3 or above 11. Here, we describe how recent insights into the structure and function of key molecules and their regulators reveal novel strategies of bacterial pH homeostasis. These insights may help us to target certain pathogens more accurately and to harness the capacities of environmental bacteria more efficiently.

The strict exchange of Na for H ions across cell membranes is a reaction carried out in almost every cell. Na /H exchangers that perform this task are physiological homodimers, and whilst the ion transporting domain is highly conserved, their dimerization differs. The Na /H exchanger NhaA from Escherichia coli has a weak dimerization interface mediated by a β-hairpin domain and with dimer retention dependent on cardiolipin. Similarly, organellar Na /H exchangers NHE6, NHE7 and NHE9 also contain β-hairpin domains and recent analysis of Equus caballus NHE9 indicated PIP lipids could bind at the dimer interface. However, structural validation of the predicted lipid-mediated oligomerization has been lacking. Here, we report cryo-EM structures of E. coli NhaA and E. caballus NHE9 in complex with cardiolipin and phosphatidylinositol-3,5-bisphosphate PI(3,5)P lipids binding at their respective dimer interfaces. We further show how the endosomal specific PI(3,5)P lipid stabilizes the NHE9 homodimer and enhances transport activity. Indeed, we show that NHE9 is active in endosomes, but not at the plasma membrane where the PI(3,5)P lipid is absent. Thus, specific lipids can regulate Na /H exchange activity by stabilizing dimerization in response to either cell specific cues or upon trafficking to their correct membrane location.

The X-ray crystal structure of NapA, a Na + /H + antiporter from Thermus thermophilus , in an active, outward-facing state is reported; comparisons to the structure of a related transporter in a low pH/inactivated, inward-facing state show the conformational changes that occur when the membrane protein moves from an inward-facing to an outward-facing state, suggesting that Na + /H + antiporters operate by a two-domain rocking bundle model. Ins and outs of a Na + /H + antiporter This manuscript reports an X-ray crystal structure of NapA, a sodium/proton antiporter from Thermus thermophilus , in an active, outward-facing state. Antiporters of this type are active in the plasma membrane of all living cells, where they help to regulate intracellular pH, sodium concentration and cell volume. Comparison of this new structure to a previously published structure of a related transporter in a low pH/inactivated, inward-facing state reveals the conformational changes that occur when the membrane protein moves from an inward-facing to an outward-facing state. Sodium/proton (Na + /H + ) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis 1 . In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets 2 . The best understood model system for Na + /H + antiport is NhaA from Escherichia coli 1 , 3 , for which both electron microscopy and crystal structures are available 4 , 5 , 6 . NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein 1 , 4 . Like many Na + /H + antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur 7 . The only reported NhaA crystal structure so far is of the low pH inactivated form 4 . Here we describe the active-state structure of a Na + /H + antiporter, NapA from Thermus thermophilus , at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding 1 , 8 , 9 directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second 3 , Na + /H + antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

The Na + /H + exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na + /Li + exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na + /H + exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity. NHA2 exchanges sodium ions for protons across cell membranes, and its activity is linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Drew et al. report the cryo-EM structure of NHA2 in detergent and nanodiscs.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions 1 . One such exception is the sperm-specific Na + /H + exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains 2 – 5 . After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca 2+ channel activation, which drives chemotaxis 2 , 6 . SLC9C1 activation is further regulated by cAMP 2 , 7 , which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH–sAC–cAMP signalling pathway in metazoa 8 , 9 , required for sperm motility and fertilization 4 . Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration—the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na + to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na + /H + exchange. Upon hyperpolarization, the S4 voltage-sensing segment of sea urchin SLC9C1 moves down, removing inhibition caused by an intracellular helix and enabling Na + /H + exchange, leading to pH-dependent activation of sAC and sperm chemotaxis.