Sodium Symporter Research Articles

Understanding fungal and plant active urea transport systems: Keys from Aspergillus nidulans and beyond

Urea is present in all ecosystems, as a result of the metabolism of different organisms and also of human activity, being the world's most common form of nitrogen fertilizer. Fungi and plants can use urea as a nitrogen source, taking it up from the environment through specialized active transport proteins. These proteins belong to a subfamily of urea/H+ symporters included in the Solute:Sodium Symporter (SSS) family of transporters. In this review we summarize the current knowledge on this group of transporters, based on our previous studies on Aspergillus nidulans UreA. We delve into its transcriptional and post-translational regulation, structure-function relationships, transport mechanism, and certain aspects of its biogenesis. Recent findings suggest that this urea transporter subfamily is more expanded than originally thought, with representatives found in organisms as diverse as Archaea and mollusks, which raises questions on evolutionary aspects. A. nidulans ureA knockout strains provide a valuable platform for expressing urea transporters from diverse sources, facilitating their characterization and functional analysis. In this context, given the close relationship between plant and fungal active urea transporters, this knowledge could serve to develop strategies to improve the efficiency of applied urea as fertilizer.

Alternating access of a bacterial homolog of neurotransmitter: sodium symporters determined from AlphaFold2 ensembles and DEER spectroscopy

Neurotransmitter:sodium symporters (NSSs) play critical roles in neural signaling by regulating neurotransmitter uptake into cells powered by sodium electrochemical gradients. Bacterial NSSs orthologs, including MhsT from Bacillus halodurans, have emerged as model systems to understand the structural motifs of alternating access in NSSs and the extent of conservation of these motifs across the family. Here, we apply a computational/experimental methodology to illuminate the conformational landscape of MhsT alternating access. Capitalizing on our recently developed method, Sampling Protein Ensembles and Conformational Heterogeneity with AlphaFold2 (SPEACH_AF), we derived clusters of MhsT models spanning the transition from inward-facing to outward-facing conformations. Systematic application of double electron-electron resonance (DEER) spectroscopy revealed ligand-dependent movements of multiple structural motifs that underpin MhsT's conformational cycle. Remarkably, comparative DEER analysis in detergent micelles and lipid nanodiscs highlights the profound effect of the environment on the energetics of conformational changes. Through experimentally derived selection of collective variables, we present a model of ion and substrate-powered transport by MhsT consistent with the conformational cycle derived from DEER. Our findings not only advance the understanding of MhsT's function but also uncover motifs of conformational dynamics conserved within the broader context of the NSS family and within the LeuT-fold class of transporters. Importantly, our methodological blueprint introduces an approach that can be applied across a diverse spectrum of transporters to describe their conformational landscapes.

Structure of the human dopamine transporter in complex with cocaine.

The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine1. As part of the neurotransmitter sodium symporter family, DAT uses the Na+ gradient across cell membranes to transport dopamine against its chemical gradient2. The transport mechanism involves both intra- and extracellular gates that control substrate access to a central site. However, the molecular intricacies of this process and the inhibitory mechanism of cocaine have remained unclear. Here, we present the molecular structure of human DAT in complex with cocaine at a resolution of 2.66 Å. Our findings reveal that DAT adopts the expected LeuT-fold, posing in an outward-open conformation with cocaine bound at the central (S1) site. Notably, while an Na+ occupies the second Na+ site (Na2), the Na1 site seems to be vacant, with the side chain of Asn82 occupying the presumed Na+ space. This structural insight elucidates the mechanism for the cocaine inhibition of human DAT and deepens our understanding of neurotransmitter transport. By shedding light on the molecular underpinnings of how cocaine acts, our study lays a foundation for the development of targeted medications to combat addiction.

Chloroplast-localized PvBASS2 regulates salt tolerance in the C4 plant seashore paspalum.

SODIUM SYMPORTER FAMILY PROTEIN 2 (BASS2) is localized within chloroplast membranes, facilitating the translocation of pyruvate and Na+ from the cytosol to the plastid, where pyruvate supports isopentenyl diphosphate (IPP) synthesis via the methylerythritol phosphate pathway in C3 plants. Nevertheless, the biological function of BASS2 in C4 plants has not been well defined. This study unveils a previously unidentified role of PvBASS2 in Na+ and pyruvate transport in seashore paspalum (Paspalum vaginatum), a halophytic C4 grass, indicating a specific cellular function within this plant species. Data showed that overexpression of PvBASS2 in seashore paspalum attenuated salt tolerance, whereas its RNAi lines exhibited enhanced salt resistance compared to wild-type plants, suggesting a negative regulatory role of PvBASS2 in seashore paspalum salt tolerance. The constitutive overexpression of PvBASS2 was also found to reduce salt tolerance in Arabidopsis. Further study revealed that PvBASS2 negatively regulates seashore paspalum salt tolerance, possibly due to elevated Na+/K+ ratio, disrupted chloroplast structure, and reduced photosynthetic efficiency following exposure to salinity. Importantly, our subsequent investigations revealed that modulation of PvBASS2 expression in seashore paspalum influenced carbon dioxide assimilation, intermediary metabolites of the tricarboxylic acid cycle, and enzymatic activities under salinity treatment, which in turn led to alterations in free amino acid concentrations. Thus, this study reveals a role for BASS2 in the C4 plant seashore paspalum and enhances our comprehension of salt stress responses in C4 plants.

Pho pictures provide powerful perspectives of phosphate importing proteins

In this issue of Structure, Schneider etal.1 report multiple structures of the low-affinity inorganic-phosphate transporter Pho90 from Saccharomyces cerevisiae. With remarkable resolution of the Divalent Anion Sodium Symporter family member, their cryo-EM studies of this fungal protein reveal new modes of sodium, substrate, and lipid binding.

Genome-Wide Identification and Expression Analysis of BrBASS Genes in Brassica rapa Reveals Their Potential Roles in Abiotic Stress Tolerance.

The bile acid sodium symporter (BASS) family plays an important role in transporting substances and coordinating plants' salt tolerance. However, the function of BASS in Brassica rapa has not yet been elucidated. In this study, eight BrBASS genes distributed on five chromosomes were identified that belonged to four subfamilies. Expression profile analysis showed that BrBASS7 was highly expressed in roots, whereas BrBASS4 was highly expressed in flowers. The promoter element analysis also identified several typical homeopathic elements involved in abiotic stress tolerance and stress-related hormonal responses. Notably, under salt stress, the expression of BrBASS2 was significantly upregulated; under osmotic stress, that of BrBASS4 increased and then decreased; and under cold stress, that of BrBASS7 generally declined. The protein-protein interaction analysis revealed that the BrBASS2 homologous gene AtBASS2 interacted with Nhd1 (N-mediated heading date-1) to alleviate salt stress in plants, while the BrBASS4 homologous gene AtBASS3 interacted with BLOS1 (biogenesis of lysosome-related organelles complex 1 subunit 1) via co-regulation with SNX1 (sorting nexin 1) to mitigate an unfavorable growing environment for roots. Further, Bra-miR396 (Bra-microRNA396) targeting BrBASS4 and BrBASS7 played a role in the plant response to osmotic and cold stress conditions, respectively. This research demonstrates that BrBASS2, BrBASS4, and BrBASS7 harbor great potential for regulating abiotic stresses. The findings will help advance the study of the functions of the BrBASS gene family.

Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT.

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.

Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+ binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a ~3-fold increase in Na+ affinity. Together, the results suggest that K+ binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NSSs.

Unique Substrate Recognition and Sodium–Substrate Binding Stoichiometry in a Bacterial Serotonin Transporter, TuriSERT

All resolved high-resolution structures of the transporters in the neurotransmitter sodium symporter (NSS) family revealed that the NSS members share common structural and mechanistic features for substrate and ion binding and transport. However, a recently reported bacterial orthologue of the human serotonin transporter (hSERT), TuriSERT, possesses a structural characteristic specific for amino acid substrate binding but does transport a biogenic amine. The unique structural feature of TuriSERT requires a novel configuration for coordinating its substrate and ions. In the present study, we characterized TuriSERT expressed in Escherichia coli cells with a fluorescent substrate by biochemical, structural, and pharmacological approaches. Substrate transport by TuriSERT requires Na+ but not Cl−. Replacement of Asp262 by asparagine renders TuriSERT Cl−-dependent. Substitutions of the corresponding Na1 residues did not alter Na+ dependence on substrate transport, whereas the mutation of a Na2 site residue led to a loss of transport activity, suggesting that Na+ binds only to the Na2 site in TuriSERT. In addition, substitutions of several residues essential for recognizing 5-hydroxytryptamine (5-HT) in hSERT had little effect on 5-HT displacement potency in transport assay for TuriSERT. In contrast, mutations of the residues that are proposed to coordinate with 5-HT in our docking model dramatically reduced 5-HT displacement. Furthermore, our results indicated that all tested antidepressants showed a weak inhibitory effect on TuriSERT. The present study demonstrated the existence of a unique substrate binding site and 1:1 stoichiometry of sodium–substrate binding in TuriSERT, a novel structural finding for the NSS transporters.

Mechanism of substrate binding and transport in BASS transporters

The bile acid sodium symporter (BASS) family transports a wide array of molecules across membranes, including bile acids in humans, and small metabolites in plants. These transporters, many of which are sodium-coupled, have been shown to use an elevator mechanism of transport, but exactly how substrate binding is coupled to sodium ion binding and transport is not clear. Here, we solve the crystal structure at 2.3 Å of a transporter from Neisseria meningitidis (ASBTNM) in complex with pantoate, a potential substrate of ASBTNM. The BASS family is characterised by two helices that cross-over in the centre of the protein in an arrangement that is intricately held together by two sodium ions. We observe that the pantoate binds, specifically, between the N-termini of two of the opposing helices in this cross-over region. During molecular dynamics simulations the pantoate remains in this position when sodium ions are present but is more mobile in their absence. Comparison of structures in the presence and absence of pantoate demonstrates that pantoate elicits a conformational change in one of the cross-over helices. This modifies the interface between the two domains that move relative to one another to elicit the elevator mechanism. These results have implications, not only for ASBTNM but for the BASS family as a whole and indeed other transporters that work through the elevator mechanism.

Mechanism of substrate binding and transport in BASS transporters.

The bile acid sodium symporter (BASS) family transports a wide array of molecules across membranes, including bile acids in humans, and small metabolites in plants. These transporters, many of which are sodium-coupled, have been shown to use an elevator mechanism of transport, but exactly how substrate binding is coupled to sodium ion binding and transport is not clear. Here, we solve the crystal structure at 2.3 Å of a transporter from Neisseria meningitidis (ASBTNM) in complex with pantoate, a potential substrate of ASBTNM. The BASS family is characterised by two helices that cross-over in the centre of the protein in an arrangement that is intricately held together by two sodium ions. We observe that the pantoate binds, specifically, between the N-termini of two of the opposing helices in this cross-over region. During molecular dynamics simulations the pantoate remains in this position when sodium ions are present but is more mobile in their absence. Comparison of structures in the presence and absence of pantoate demonstrates that pantoate elicits a conformational change in one of the cross-over helices. This modifies the interface between the two domains that move relative to one another to elicit the elevator mechanism. These results have implications, not only for ASBTNM but for the BASS family as a whole and indeed other transporters that work through the elevator mechanism.

Carbazole derivatives as promising competitive and allosteric inhibitors of human serotonin transporter: computational pharmacology

The human serotonin transporters (hSERTs) are neurotransmitter sodium symporters of the aminergic G protein-coupled receptors, regulating the synaptic serotonin and neuropharmacological processes related to neuropsychiatric disorders, notably, depression. Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine and (S)-citalopram are competitive inhibitors of hSERTs and are commonly the first-line medications for major depressive disorder (MDD). However, treatment-resistance and unpleasant aftereffects constitute their clinical drawbacks. Interestingly, vilazodone emerged with polypharmacological (competitive and allosteric) inhibitions on hSERTs, amenable to improved efficacy. However, its application usually warrants adjuvant/combination therapy, another subject of critical adverse events. Thus, the discovery of alternatives with polypharmacological potentials (one-drug-multiple-target) and improved safety remains essential. In this study, carbazole analogues from chemical libraries were explored using docking and molecular dynamics (MD) simulation. Selectively, two IBScreen ligands, STOCK3S-30866 and STOCK1N-37454 predictively bound to the active pockets and expanded boundaries (extracellular vestibules) of the hSERTs more potently than vilazodone and (S)-citalopram. For instance, the two ligands showed docking scores of −9.52 and −9.59 kcal/mol and MM-GBSA scores of −92.96 and −65.66 kcal/mol respectively compared to vilazodone’s respective scores of −7.828 and −59.27 against the central active site of the hSERT (PDB 7LWD). Similarly, the two ligands also docked to the allosteric pocket (PDB 5I73) with scores of −8.15 and −8.40 kcal/mol and MM-GBSA of −96.14 and −68.46 kcal/mol whereas (S)-citalopram has −6.90 and −69.39 kcal/mol respectively. The ligands also conferred conformational stability on the receptors during 100 ns MD simulations and displayed interesting ADMET profiles, representing promising hSERT modulators for MDD upon experimental validation. Communicated by Ramaswamy H. Sarma

Structural basis of GABA reuptake inhibition.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1), a member of the neurotransmitter sodium symporter (NSS) family, regulates neuronal excitation by clearing the synaptic cleft of the main inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating GABA levels in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we present the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor, the anticonvulsant tiagabine. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine. Tiagabine's pharmacodynamic profile, confirmed by our experimental data, suggests initial binding to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step induced-fit mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators.

Crystallographic Characterization of Sodium Ions in a Bacterial Leucine/Sodium Symporter

Na+ is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na+ in protein structures is crucial for gaining a deeper understanding of protein function in a physiological context. LeuT, a bacterial homolog of the neurotransmitter:sodium symporter family, uses the Na+ gradient to power the uptake of amino acids into cells and has been used as a paradigm for the study of Na+-dependent transport systems. We have devised a low-energy multi-crystal approach for characterizing low-Z (Z ≤ 20) anomalous scattering ions such as Na+, Mg2+, K+, and Ca2+ by combining Bijvoet-difference Fourier syntheses for ion detection and f” refinements for ion speciation. Using the approach, we experimentally identify two Na+ bound near the central leucine binding site in LeuT. Using LeuT microcrystals, we also demonstrate that Na+ may be depleted to study conformational changes in the LeuT transport cycle.

Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [3H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (Kd) for [3H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.

Pyruvate transporter BnaBASS2 impacts seed oil accumulation in Brassica napus.

Bile acid: sodium symporter family protein 2 (BASS2) is a sodium-dependent pyruvate transporter, which transports pyruvate from cytosol into plastid in plants. In this study, we investigated the function of chloroplast envelope membrane-localized BnaBASS2 in seed metabolism and seed oil accumulation of Brassica napus (B. napus). Four BASS2 genes were identified in the genome of B. napus. BnaA05.BASS2 was overexpressed while BnaA05.BASS2 and BnaC04.BASS2-1 were mutated by CRISPR in B. napus. Metabolite analysis revealed that the manipulation of BnaBASS2 caused significant changes in glycolysis-, fatty acid synthesis-, and energy-related metabolites in the chloroplasts of 31 day-after-flowering (DAF) seeds. The analysis of fatty acids and lipids in developing seeds showed that BnaBASS2 could affect lipid metabolism and oil accumulation in developing seeds. Moreover, the overexpression (OE) of BnaA05.BASS2 could promote the expression level of multiple genes involved in the synthesis of oil and the formation of oil body during seed development. Disruption of BnaA05.BASS2 and BnaC04.BASS2-1 resulted in decreasing the seed oil content (SOC) by 2.8%-5.0%, while OE of BnaA05.BASS2 significantly promoted the SOC by 1.4%-3.4%. Together, our results suggest that BnaBASS2 is a potential target gene for breeding B. napus with high SOC.

A New Method for Rapid Subcellular Localization and Gene Function Analysis in Cotton Based on Barley Stripe Mosaic Virus

The difficulty of genetic transformation has restricted research on functional genomics in cotton. Thus, a rapid and efficient method for gene overexpression that does not rely on genetic transformation is needed. Virus-based vectors offer a reasonable alternative for protein expression, as viruses can infect the host systemically to achieve expression and replication without transgene integration. Previously, a novel four-component barley stripe mosaic virus (BSMV) was reported to overexpress large fragments of target genes in plants over a long period of time, which greatly simplified the study of gene overexpression. However, whether this system can infect cotton and stably overexpress target genes has not yet been studied. In this study, we verified that this new BSMV system can infect cotton through seed imbibition and systemically overexpress large fragments of genes (up to 2340 bp) in cotton. The target gene that was fused with GFP was expressed at a high level in the roots, stems, and cotyledons of cotton seedlings, and stable fluorescence signals were detected in the cotton roots and leaves even after 4 weeks. Based on the BSMV overexpression system, the subcellular localization marker line of endogenous proteins localized in the nucleus, endoplasmic reticulum, plasma membrane, Golgi body, mitochondria, peroxisomes, tonoplast, and plastids were quickly established. The overexpression of a cotton Bile Acid Sodium Symporter GhBASS5 using the BSMV system indicated that GhBASS5 negatively regulated salt tolerance in cotton by transporting Na+ from underground to the shoots. Furthermore, multiple proteins were co-delivered, enabling co-localization and the study of protein–protein interactions through co-transformation. We also confirmed that the BSMV system can be used to conduct DNA-free gene editing in cotton by delivering split-SpCas9/sgRNA. Ultimately, the present work demonstrated that this BSMV system could be used as an efficient overexpression system for future cotton gene function research.

Structural basis of ion – substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY’s conformational selection mechanism is a result of the sodium-dependent formation of the substrate binding site.

The dopamine transporter antiports potassium to increase the uptake of dopamine

The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na+ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K+, but investigations of K+-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the Drosophila- and human dopamine transporters are inhibited by K+, and the conformational dynamics of the Drosophila dopamine transporter in K+ are divergent from the apo- and Na+-states. Furthermore, we find that K+ increases dopamine uptake by the Drosophila dopamine transporter in liposomes, and visualize Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other transporters in this pharmacologically important family.

Transcriptome Analysis of Crassostrea sikamea (♀) × Crassostrea gigas (♂) Hybrids Under Hypoxia in Occluded Water

Hypoxia is considered to be one of the key factors affecting the survival of ocean organisms, it is necessary to parse the molecular processes involved in response to hypoxia. As a potential breeding species, the hybrid of Crassostrea sikamea (♀) × Crassostrea gigas (♂) shows valuable heterosis in survival and growth traits. Thus, RNA de novo was deployed in this study to analyze the molecular processes in the hybrids under hypoxia stress. The hybrids were cultured in occluded water, then the dissolved oxygen was gradually consumed by oysters, and the gill tissue of hybrids was sampled at the very beginning and the lowest respiration point in the experiment. In the current study, 901 significant differentially expressed genes (DEGs) were identified under hypoxia compared to normoxia, among which 432 DEGs were downregulated, and the other 469 DEGs were upregulated. A total of 27 GO terms were significantly enriched, such as an integral component of membrane, extracellular region, immune response, tumor necrosis factor receptor binding, and neurotransmitter: sodium symporter activity. Besides, 19 KEGG pathways were significantly enriched, such as apoptosis, Th1 and Th2 cell differentiation, complement, and coagulation cascades, antigen processing and presentation, notch signaling pathway, and cytokine–cytokine receptor interaction. The current results showed that the TRAIL genes were downregulated, but the HSP70 and LIGHT genes were upregulated, which indicated the inhibition of Apoptosis, and the activity of innate immunity in oysters under hypoxia. This study provides preliminary insight into the molecular response to hypoxia in the gill of hybrids.