Membraneless channels sieve cations in ammonia-oxidizing marine archaea.

Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle1,2. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. Biochemical analyses showed strong ammonium binding by the cell surface, which was lost after S-layer disassembly. Sensitive bioinformatic analyses identified similar S-layers in many ammonia-oxidizing archaea, with conserved sequence and structural characteristics. Moreover, molecular simulations and structure determination of ammonium-enriched specimens enabled us to examine the cation-binding properties of the S-layer, revealing how it concentrates ammonium ions on its cell-facing side, effectively acting as a multichannel sieve on the cell membrane. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.

Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Dilation of ion selectivity filters in cation channels.

Ion channels establish the voltage gradient across cellular membranes by providing aqueous pathways for ions to selectively diffuse down their concentration gradients. The selectivity of any given channel for its favored ions has conventionally been viewed as a stable property, and in many cation channels, it is determined by an ion-selectivity filter within the external end of the ion-permeation pathway. In several instances, including voltage-activated K+ (Kv) channels, ATP-activated P2X receptor channels, and transient receptor potential (TRP) channels, the ion-permeation pathways have been proposed to dilate in response to persistent activation, dynamically altering ion permeation. Here, we discuss evidence for dynamic ion selectivity, examples where ion selectivity filters exhibit structural plasticity, and opportunities to fill gaps in our current understanding.

Direct evidence for phosphorus limitation on Amazon forest productivity.

The productivity of rainforests growing on highly weathered tropical soils is expected to be limited by phosphorus availability1. Yet, controlled fertilization experiments have been unable to demonstrate a dominant role for phosphorus in controlling tropical forest net primary productivity. Recent syntheses have demonstrated that responses to nitrogen addition are as large as to phosphorus2, and adaptations to low phosphorus availability appear to enable net primary productivity to be maintained across major soil phosphorus gradients3. Thus, the extent to which phosphorus availability limits tropical forest productivity is highly uncertain. The majority of the Amazonia, however, is characterized by soils that are more depleted in phosphorus than those in which most tropical fertilization experiments have taken place2. Thus, we established a phosphorus, nitrogen and base cation addition experiment in an old growth Amazon rainforest, with a low soil phosphorus content that is representative of approximately 60% of the Amazon basin. Here we show that net primary productivity increased exclusively with phosphorus addition. After 2 years, strong responses were observed in fine root (+29%) and canopy productivity (+19%), but not stem growth. The direct evidence of phosphorus limitation of net primary productivity suggests that phosphorus availability may restrict Amazon forest responses to CO2 fertilization4, with major implications for future carbon sequestration and forest resilience to climate change.

A wheat resistosome defines common principles of immune receptor channels.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) detect pathogen effectors to trigger immune responses1. Indirect recognition of a pathogen effector by the dicotyledonous Arabidopsis thaliana coiled-coil domain containing NLR (CNL) ZAR1 induces the formation of a large hetero-oligomeric protein complex, termed the ZAR1 resistosome, which functions as a calcium channel required for ZAR1-mediated immunity2-4. Whether the resistosome and channel activities are conserved among plant CNLs remains unknown. Here we report the cryo-electron microscopy structure of the wheat CNL Sr355 in complex with the effector AvrSr356 of the wheat stem rust pathogen. Direct effector binding to the leucine-rich repeats of Sr35 results in the formation of a pentameric Sr35-AvrSr35 complex, which we term the Sr35 resistosome. Wheat Sr35 and Arabidopsis ZAR1 resistosomes bear striking structural similarities, including an arginine cluster in the leucine-rich repeats domain not previously recognized as conserved, which co-occurs and forms intramolecular interactions with the 'EDVID' motif in the coiled-coil domain. Electrophysiological measurements show that the Sr35 resistosome exhibits non-selective cation channel activity. These structural insights allowed us to generate new variants of closely related wheat and barley orphan NLRs that recognize AvrSr35. Our data support the evolutionary conservation of CNL resistosomes in plants and demonstrate proof of principle for structure-based engineering of NLRs for crop improvement.

Electrochemical control of cell and tissue polarity.

Localized ion fluxes at the plasma membrane provide electrochemical gradients at the cell surface that contribute to cell polarization, migration, and division. Ion transporters, local pH gradients, membrane potential, and organization are emerging as important factors in cell polarization mechanisms. The power of electrochemical effects is illustrated by the ability of exogenous electric fields to redirect polarization in cells ranging from bacteria, fungi, and amoebas to keratocytes and neurons. Electric fields normally surround cells and tissues and thus have been proposed to guide cell polarity in development, cancer, and wound healing. Recent studies on electric field responses in model systems and development of new biosensors provide new avenues to dissect molecular mechanisms. Here, we review recent advances that bring molecular understanding of how electrochemistry contributes to cell polarity in various contexts.

ATP-elicited Cation Fluxes Promote Volume-regulated Anion Channel LRRC8/VRAC Transport cGAMP for Antitumor Immunity.

The cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway is instrumental to antitumor immunity, yet the underlying molecular and cellular mechanisms are complex and still unfolding. A new paradigm suggests that cancer cells' cGAS-synthesized cGAMP can be transferred to tumor-infiltrating immune cells, eliciting STING-dependent IFN-ß response for antitumor immunity. Nevertheless, how the tumor microenvironment may shape this process remains unclear. In this study, we found that extracellular ATP, an immune regulatory molecule widely present in the tumor microenvironment, can potentiate cGAMP transfer, thereby boosting the STING signaling and IFN-ß response in murine macrophages and fibroblasts. Notably, genetic ablation or chemical inhibition of murine volume-regulation anion channel LRRC8/volume-regulated anion channel (VRAC), a recently identified cGAMP transporter, abolished ATP-potentiated cGAMP transfer and STING-dependent IFN-ß response, revealing a crucial role of LRRC8/VRAC in the cross-talk of extracellular ATP and cGAMP. Mechanistically, ATP activation of the P2X family receptors triggered Ca2+ influx and K+ efflux, promoting reactive oxygen species production. Moreover, ATP-evoked K+ efflux alleviated the phosphorylation of VRAC's obligate subunit LRRC8A/SWELL1 on S174. Mutagenesis studies indicated that the phosphorylation of S174 on LRRC8A could act as a checkpoint for VRAC in the steady state and a rheostat of ATP responsiveness. In an MC38-transplanted tumor model, systemically blocking CD39 and ENPP1, hydroxylases of extracellular ATP and cGAMP, respectively, elevated antitumor NK, NKT, and CD8+ T cell responses and restrained tumor growth in mice. Altogether, this study establishes a crucial role of ATP in facilitating LRRC8/VRAC transport cGAMP in the tumor microenvironment and provides new insight into harnessing cGAMP transfer for antitumor immunity.

Antithetic effects of agonists and antagonists on the structural fluctuations of TRPV1 channel.

Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity (ΔCp) model [D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U.S.A. 108, 19492-19497 (2011).] has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (agonist, 1,000 times hotter than capsaicin) and capsazepine (antagonist) by high-speed atomic force microscopy. We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.

Several orphan solute carriers functionally identified as organic cation transporters: Substrates specificity compared with known cation transporters.

Organic cations comprise a significant part of medically relevant drugs and endogenous substances. Such substances need organic cation transporters for efficient transfer via cell membranes. However, the membrane transporters of most natural or synthetic organic cations are still unknown. To identify these transporters, genes of 10 known OCTs and 18 orphan solute carriers (SLC) were overexpressed in HEK293 cells and characterized concerning their transport activities with a broad spectrum of low molecular weight substances emphasizing organic cations. Several SLC35 transporters and SLC38A10 significantly enhanced the transport of numerous relatively hydrophobic organic cations. Significant organic cation transport activities have been found in gene families classified as transporters of other substance classes. For instance, SLC35G3 and SLC38A10 significantly accelerated the uptake of several cations, such as clonidine, 3,4-methylenedioxymethamphetamine, and nicotine, which are known as substrates of a thus far genetically unidentified proton/organic cation antiporter. The transporters SLC35G4 and SLC35F5 stood out by their significantly increased choline uptake, and several other SLC transported choline together with a broader spectrum of organic cations. Overall, there are many more polyspecific organic cation transporters than previously estimated. Several transporters had one predominant substrate but accepted some other cationic substrates, and others showed no particular preference for one substrate but transported several organic cations. The role of these transporters in biology and drug therapy remains to be elucidated.

Distinct roles of the major binding residues in the cation-binding pocket of the melibiose transporter MelB.

Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.

Endo-Lysosomal Cation Channels and Infectious Diseases.

Among the infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi, the most prevalent ones today are malaria, tuberculosis, influenza, HIV/AIDS, Ebola, dengue fever, and methicillin-resistant Staphylococcus aureus (MRSA) infection, and most recently Covid-19 (SARS-CoV2). Others with a rather devastating history and high fatality rates such as plague, cholera, or typhus seem less threatening today but have not been eradicated, and with a declining efficacy of current antibiotics they ought to be watched carefully. Another emerging issue in this context is health-care associated infection. About 100,000 hospitalized patients in the USA ( www.cdc.gov ) and 33,000 in Europe ( https://www.ecdc.europa.eu ) die each year as a direct consequence of an infection caused by bacteria resistant to antibiotics. Among viral infections, influenza is responsible for about 3-5 million cases of severe illness, and about 250,000 to 500,000 deaths annually ( www.who.int ). About 37 million people are currently living with HIV infection and about one million die from it each year. Coronaviruses such as MERS-CoV, SARS-CoV, but in particular the recent outbreak of Covid-19 (caused by SARS-CoV2) have resulted in large numbers of infections worldwide with an estimated several hundred thousand deaths (anticipated fatality rate: <5%). With a comparatively low mortality rate dengue virus causes between 50 and 100 million infections every year, leading to 50,000 deaths. In contrast, Ebola virus is the causative agent for one of the deadliest viral diseases. The Ebola outbreak in West Africa in 2014 is considered the largest outbreak in history with more than 11,000 deaths. Many of the deadliest pathogens such as Ebola virus, influenza virus, mycobacterium tuberculosis, dengue virus, and cholera exploit the endo-lysosomal trafficking system of host cells for penetration into the cytosol and replication. Defects in endo-lysosomal maturation, trafficking, fusion, or pH homeostasis can efficiently reduce the cytotoxicity caused by these pathogens. Most of these functions critically depend on endo-lysosomal membrane proteins such as transporters and ion channels. In particular, cation channels such as the mucolipins (TRPMLs) or the two-pore channels (TPCs) are involved in all of these aspects of endo-lysosomal integrity. In this review we will discuss the correlations between pathogen toxicity and endo-lysosomal cation channel function, and their potential as drug targets for infectious disease therapy.

Structure of the human cation-chloride cotransport KCC1 in an outward-open state.

Cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concentration, and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+-Cl- cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid-polyamine-organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helices. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small molecule compounds.

Elevated intracellular Na+ and osmolarity stimulate catalytic activity of the ubiquitin ligase Nedd4-2.

Regulation of catalytic activity of E3 ubiquitin ligases is critical for their cellular functions. We identified an unexpected mode of regulation of E3 catalytic activity by ions and osmolarity; enzymatic activity of the HECT family E3 Nedd4-2/Nedd4L is enhanced by increased intracellular Na+ ([Na+]i) and by hyperosmolarity. This stimulated activity is mediated by activation of p38-MAPK and is inhibited by WNKs. Moreover, protease (Furin)-mediated activation of the epithelial Na+ channel ENaC (a bona fide Nedd4-2 substrate), which leads to increased [Na+]i and osmolarity, results in enhanced Nedd4-2 catalytic activity. This enhancement is inhibited by a Furin inhibitor, by a protease-resistant ENaC mutant, or by treatment with the ENaC inhibitor amiloride. Moreover, WNK inhibition, which stimulates catalytic activity of Nedd4-2, leads to reduced levels of cell-surface ENaC and reduced channel activity. ENaC activity does not affect Nedd4-2:ENaC binding. Therefore, these results demonstrate activation of a ubiquitin ligase by Na+ and osmotic changes. Importantly, they reveal a negative feedback loop in which active ENaC leads to stimulation of catalytic activity of its own suppressor, Nedd4-2, to protect cells from excessive Na+ loading and hyperosmotic stress and to protect the animal from hypertension.

Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules.

Protecting the Achilles heel: three FolE_I-type GTP-cyclohydrolases needed for full growth of metal-resistant Cupriavidus metallidurans under a variety of conditions.

In Cupriavidus metallidurans and other bacteria, biosynthesis of the essential biochemical cofactor tetrahydrofolate (THF) initiates from guanosine triphosphate (GTP). This step is catalyzed by FolE_I-type GTP cyclohydrolases, which are either zinc-dependent FolE_IA-type or metal-promiscuous FolE_IB-type enzymes. As THF is also essential for GTP biosynthesis, GTP and THF synthesis form a cooperative cycle, which may be influenced by the cellular homeostasis of zinc and other metal cations. Metal-resistant C. metallidurans harbors one FolE_IA-type and two FolE_IB-type enzymes. All three proteins were produced in Escherichia coli. FolE_IA was indeed zinc dependent and the two FolE_IB enzymes metal-promiscuous GTP cyclohydrolases in vitro, the latter, for example, functioning with iron, manganese, or cobalt. Single and double mutants of C. metallidurans with deletions in the folE_I genes were constructed to analyze the contribution of the individual FolE_I-type enzymes under various conditions. FolE_IA was required in the presence of cadmium, hydrogen peroxide, metal chelators, and under general metal starvation conditions. FolE_IB1 was important when zinc uptake was impaired in cells without the zinc importer ZupT (ZIP family) and in the presence of trimethoprim, an inhibitor of THF biosynthesis. FolE_IB2 was needed under conditions of low zinc and cobalt but high magnesium availability. Together, these data demonstrate that C. metallidurans requires all three enzymes to allow efficient growth under a variety of conditions.IMPORTANCETetrahydrofolate (THF) is an important cofactor in microbial biochemistry. This "Achilles heel" of metabolism has been exploited by anti-metabolites and antibiotics such as sulfonamide and trimethoprim. Since THF is essential for the synthesis of guanosine triphosphate (GTP) and THF biosynthesis starts from GTP, synthesis of both compounds forms a cooperative cycle. The first step of THF synthesis by GTP cyclohydrolases (FolEs) is metal dependent and catalyzed by zinc- or metal-promiscuous enzymes, so that the cooperative THF and GTP synthesis cycle may be influenced by the homeostasis of several metal cations, especially that of zinc. The metal-resistant bacterium C. metallidurans needs three FolEs to grow in environments with both high and low zinc and cadmium content. Consequently, bacterial metal homeostasis is required to guarantee THF biosynthesis.

Stretch response of the mechano-gated channel TMEM63A in membrane patches and single cells.

The recently discovered ion channel TMEM63A has biophysical features distinctive for mechano-gated cation channels, activating at high pressures with slow kinetics while not inactivating. However, some biophysical properties are less clear, including no information on its function in whole cells. The aim of this study is to expand the TMEM63A biophysical characterization and examine the function in whole cells. Piezo1-knockout HEK293T cells were cotransfected with human TMEM63A and green fluorescent protein (GFP), and macroscopic currents in cell-attached patches were recorded by high-speed pressure clamp at holding voltages from -120 to -20 mV with 0-100 mmHg patch suction for 1 s. HEK293 cells cotransfected with TMEM63A and GCaMP5 were seeded onto polydimethylsiloxane (PDMS) membrane, and the response to 3-12 s of 1%-15% whole cell isotropic (equi-biaxial) stretch induced by an IsoStretcher was measured by the change in intracellular calcium ([Ca2+]i) and presented as (ΔF/F0 > 1). Increasing patch pressures activated TMEM63A currents with accelerating activation kinetics and current amplitudes that were pressure dependent but voltage independent. TMEM63A currents were plateaued within 2 s, recovered quickly, and were sensitive to Gd3+. In whole cells stretched on flexible membranes, radial stretch increased the [Ca2+]i responses in a larger proportion of cells cotransfected with TMEM63A and GCaMP5 than GCaMP5-only controls. TMEM63A currents are force activated and voltage insensitive, have a high threshold for pressure activation with slow activation and deactivation, and lack inactivation over 5 s. TMEM63A has the net polarity and kinetics that would depolarize plasma membranes and increase inward currents, contributing to a sustained [Ca2+]i increase in response to high stretch.NEW & NOTEWORTHY TMEM63A has biophysical features distinctive for mechano-gated cation channels, but some properties are less clear, including no functional information in whole cells. We report that pressure-dependent yet voltage-independent TMEM63A currents in cell membrane patches correlated with cell size. In addition, radial stretch of whole cells on flexible membranes increased the [Ca2+]i responses more in TMEM63A-transfected cells. Inward TMEM63A currents in response to high stretch can depolarize plasma membranes and contribute to a sustained [Ca2+]i increase.

A splicing-dependent ER retention signal regulates surface expression of the mechanosensitive TMEM63B cation channel.

TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon 4 in TMEM63B. The short variant lacking exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.

The yeast Gdt1 protein mediates the exchange of H+ for Ca2+ and Mn2+ influencing the Golgi pH.

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photosynthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca2+ or Mn2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H+ ions are transported in exchange for Ca2+ and Mn2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca2+, Mn2+ or H+ gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H+ ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H+ transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts.

Genome-wide characterization of the CPA gene family in potato and a preliminary functional analysis of its role in NaCl tolerance.

BACKGROUND: The cation/proton antiporter (CPA) superfamily plays a crucial role in regulating ion homeostasis and pH in plant cells, contributing to stress resistance. However, in potato (Solanum tuberosum L.), systematic identification and analysis of CPA genes are lacking. RESULTS: A total of 33 StCPA members were identified and classified into StNHX (n = 7), StKEA (n = 6), and StCHX (n = 20) subfamilies. StCHX owned the highest number of conserved motifs, followed by StKEA and StNHX. The StNHX and StKEA subfamilies owned more exons than StCHX. NaCl stress induced the differentially expression of 19 genes in roots or leaves, among which StCHX14 and StCHX16 were specifically induced in leaves, while StCHX2 and StCHX19 were specifically expressed in the roots. A total of 11 strongly responded genes were further verified by qPCR. Six CPA family members, StNHX1, StNHX2, StNHX3, StNHX5, StNHX6 and StCHX19, were proved to transport Na+ through yeast complementation experiments. CONCLUSIONS: This study provides comprehensive insights into StCPAs and their response to NaCl stress, facilitating further functional characterization.

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism.

The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.