Monocarboxylate Transporters 1 and 2 Are Responsible for L-Lactate Uptake in Differentiated Human Neuroblastoma SH-SY5Y Cells.

L-Lactate transport via monocarboxylate transporters (MCTs) in the central nervous system, represented by the astrocyte-neuron lactate shuttle (ANLS), is crucial for the maintenance of brain functions, including memory formation. Previously, we have reported that MCT1 contributes to L-lactate transport in normal human astrocytes. Therefore, in this study, we aimed to identify transporters that contribute to L-lactate transport in human neurons. SH-SY5Y cells, which are used as a model for human neurons, were differentiated using all-trans-retinoic acid. L-Lactate uptake was measured using radiolabeled L-lactate, and the expression of MCT proteins was confirmed Western blotting. L-Lactate transport was pH-dependent and saturated at high concentrations. Kinetic analysis suggested that L-lactate uptake was biphasic. Furthermore, MCT1, 2 selective inhibitors inhibited L-lactate transport. In addition, the expression of MCT1 and 2 proteins, but not MCT4, was confirmed. In this study, we demonstrated that MCT1 and 2 are major contributors to L-lactate transport in differentiated human neuroblastoma SH-SY5Y cells from the viewpoint of kinetic analysis. These results lead to a better understanding of ANLS in humans, and further exploration of the factors that can promote MCT1 and 2 functions is required.

Characterisation of anhydro-sialic acid transporters from mucosa-associated bacteria.

Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise Sia for growth and/or cell surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-Sia forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

Cryo-EM structures of the human NaS1 and NaDC1 transporters revealed the elevator transport and allosteric regulation mechanism.

The solute carrier 13 (SLC13) family comprises electrogenic sodium ion-coupled anion cotransporters, segregating into sodium ion-sulfate cotransporters (NaSs) and sodium ion-di- and-tricarboxylate cotransporters (NaDCs). NaS1 and NaDC1 regulate sulfate homeostasis and oxidative metabolism, respectively. NaS1 deficiency affects murine growth and fertility, while NaDC1 affects urinary citrate and calcium nephrolithiasis. Despite their importance, the mechanisms of substrate recognition and transport remain insufficiently characterized. In this study, we determined the cryo-electron microscopy structures of human NaS1, capturing inward-facing and combined inward-facing/outward-facing conformations within a dimer both in apo and sulfate-bound states. In addition, we elucidated NaDC1's outward-facing conformation, encompassing apo, citrate-bound, and N-(p-amylcinnamoyl) anthranilic acid (ACA) inhibitor-bound states. Structural scrutiny illuminates a detailed elevator mechanism driving conformational changes. Notably, the ACA inhibitor unexpectedly binds primarily anchored by transmembrane 2 (TM2), Loop 10, TM11, and TM6a proximate to the cytosolic membrane. Our findings provide crucial insights into SLC13 transport mechanisms, paving the way for future drug design.

Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

Expression of the monocarboxylate transporter MCT1 is required for virus-specific mouse CD8+ T cell memory development.

Lactate-proton symporter monocarboxylate transporter 1 (MCT1) facilitates lactic acid export from T cells. Here, we report that MCT1 is mandatory for the development of virus-specific CD8+ T cell memory. MCT1-deficient T cells were exposed to acute pneumovirus (pneumonia virus of mice, PVM) or persistent γ-herpesvirus (Murid herpesvirus 4, MuHV-4) infection. MCT1 was required for the expansion of virus-specific CD8+ T cells and the control of virus replication in the acute phase of infection. This situation prevented the subsequent development of virus-specific T cell memory, a necessary step in containing virus reactivation during γ-herpesvirus latency. Instead, persistent active infection drove virus-specific CD8+ T cells toward functional exhaustion, a phenotype typically seen in chronic viral infections. Mechanistically, MCT1 deficiency sequentially impaired lactic acid efflux from activated CD8+ T cells, caused an intracellular acidification inhibiting glycolysis, disrupted nucleotide synthesis in the upstream pentose phosphate pathway, and halted cell proliferation which, ultimately, promoted functional CD8+ T cell exhaustion instead of memory development. Taken together, our data demonstrate that MCT1 expression is mandatory for inducing T cell memory and controlling viral infection by CD8+ T cells.

The expression system influences stability, maturation efficiency, and oligomeric properties of the potassium-chloride co-transporter KCC2.

The neuron-specific K+/Cl- co-transporter 2, KCC2, which is critical for brain development, regulates γ-aminobutyric acid-dependent inhibitory neurotransmission. Consistent with its function, mutations in KCC2 are linked to neurodevelopmental disorders, including epilepsy, schizophrenia, and autism. KCC2 possesses 12 transmembrane spans and forms an intertwined dimer. Based on its complex architecture and function, reduced cell surface expression and/or activity have been reported when select disease-associated mutations are present in the gene encoding the protein, SLC12A5. These data suggest that KCC2 might be inherently unstable, as seen for other complex polytopic ion channels, thus making it susceptible to cellular quality control pathways that degrade misfolded proteins. To test these hypotheses, we examined KCC2 stability and/or maturation in five model systems: yeast, HEK293 cells, primary rat neurons, and rat and human brain synaptosomes. Although studies in yeast revealed that KCC2 is selected for endoplasmic reticulum-associated degradation (ERAD), experiments in HEK293 cells supported a more subtle role for ERAD in maintaining steady-state levels of KCC2. Nevertheless, this system allowed for an analysis of KCC2 glycosylation in the ER and Golgi, which serves as a read-out for transport through the secretory pathway. In turn, KCC2 was remarkably stable in primary rat neurons, suggesting that KCC2 folds efficiently in more native systems. Consistent with these data, the mature glycosylated form of KCC2 was abundant in primary rat neurons as well as in rat and human brain. Together, this work details the first insights into the influence that the cellular and membrane environments have on several fundamental KCC2 properties, acknowledges the advantages and disadvantages of each system, and helps set the stage for future experiments to assess KCC2 in a normal or disease setting.

Cryptochrome PtCPF1 regulates high temperature acclimation of marine diatoms through coordination of iron and phosphorus uptake.

Increasing ocean temperatures threaten the productivity and species composition of marine diatoms. High temperature response and regulation are important for the acclimation of marine diatoms to such environments. However, the molecular mechanisms behind their acclimation to high temperature are still largely unknown. In this study, the abundance of PtCPF1 homologs (a member of the cryptochrome-photolyase family in the model diatom Phaeodactylum tricornutum) transcripts in marine phytoplankton is shown to increase with rising temperature based on Tara Oceans datasets. Moreover, the expression of PtCPF1 in P. tricornutum at high temperature (26 °C) was much higher than that at optimum temperature (20 °C). Deletion of PtCPF1 in P. tricornutum disrupted the expression of genes encoding two phytotransferrins (ISIP2A and ISIP1) and two Na+/P co-transporters (PHATRDRAFT_47667 and PHATRDRAFT_40433) at 26 °C. This further impacted the uptake of Fe and P, and eventually caused the arrest of cell division. Gene expression, Fe and P uptake, and cell division were restored by rescue with the native PtCPF1 gene. Furthermore, PtCPF1 interacts with two putative transcription factors (BolA and TF IIA) that potentially regulate the expression of genes encoding phytotransferrins and Na+/P co-transporters. To the best of our knowledge, this is the first study to reveal PtCPF1 as an essential regulator in the acclimation of marine diatoms to high temperature through the coordination of Fe and P uptake. Therefore, these findings help elucidate how marine diatoms acclimate to high temperature.

Basigin mediation of Plasmodium falciparum red blood cell invasion does not require its transmembrane domain or interaction with monocarboxylate transporter 1.

Plasmodium falciparum invasion of the red blood cell is reliant upon the essential interaction of PfRh5 with the host receptor protein basigin. Basigin exists as part of one or more multiprotein complexes, most notably through interaction with the monocarboxylate transporter MCT1. However, the potential requirement for basigin association with MCT1 and the wider role of basigin host membrane context and lateral protein associations during merozoite invasion has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate the ability to uncouple basigin ectodomain presentation from its transmembrane domain-mediated interactions, including with MCT1. Merozoite invasion of reticulocytes is unaffected by disruption of basigin-MCT1 interaction and by removal or replacement of the basigin transmembrane helix. Therefore, presentation of the basigin ectodomain at the red blood cell surface, independent of its native association with MCT1 or other interactions mediated by the transmembrane domain, is sufficient to facilitate merozoite invasion.

Sodium-iodide symporter and its related solute carriers in thyroid cancer.

The solute carrier (SLC) family is a large group of membrane transport proteins. Their dysfunction plays an important role in the pathogenesis of thyroid cancer. The most well-known SLC is the sodium-iodide symporter (NIS), also known as sodium/iodide co-transporter or solute carrier family 5 member 5 (SLC5A5) in thyroid cancer. The dysregulation of NIS in thyroid cancer is well documented. The role of NIS in the uptake of iodide is critical in the treatment of thyroid cancer, radioactive iodide (RAI) therapy in particular. In addition to NIS, other SLC members may affect the autophagy, proliferation, and apoptosis of thyroid cancer cells, indicating that an alteration in SLC members may affect different cellular events in the evolution of thyroid cancer. The expression of the SLC members may impact the uptake of chemicals by the thyroid, suggesting that targeting SLC members may be a promising therapeutic strategy in thyroid cancer.

Mechanism of multi-organ compensation under different iodine intake in pregnant rats: results from a repeated-measures study of iodine metabolism.

PURPOSE: This study aimed to explore the differences in iodine metabolism and expression of NIS and Pendrin in pregnant rats under different iodine nutritional status. METHODS: Female Wistar rats were divided into four groups: low iodine (LI), normal iodine (NI), ten fold high iodine (10HI), and fifty fold high iodine (50HI). The intervention began after one week of adaptive feeding. Iodine metabolism experiments were performed beginning on the 15th day of pregnancy. 24-h iodine intake and excretion were calculated. The concentrations of iodine in urine, fecal, thyroid, and placenta were measured by ICP-MS. PCR and Western Blot were used to detect the mRNA levels and cell membrane protein of sodium/iodide symporter (NIS) and Pendrin in the small intestine, thyroid, kidney, and placenta. RESULTS: Fecal iodine excretion (FIE) and urinary iodine excretion (UIE) in the 50HI group were significantly higher than those in the NI group (P < 0.05). The NIS protein and mRNA in the kidney and small intestine have an upward trend in iodine deficiency and a downward trend in iodine excess. Thyroid and placental iodine storage in the 50HI group were significantly higher than those in the NI group (P < 0.05). NIS, Pendrin protein, and mRNA in the thyroid and placenta tend to increase when iodine is deficient and decrease when there is excess. CONCLUSION: Iodine excretion and iodine stores in the placenta and thyroid gland are positively correlated with iodine intake. NIS and Pendrin are also regulated by iodine intake.

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.

Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

CMTM 6 promotes the development of thyroid cancer by inhibiting NIS activity through activating the MAPK signaling pathway.

Thyroid cancer is the most common type of endocrine cancer. Chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing 6 (CMTM6) is recognized as one of its potential immunotherapy targets. The purpose of this study was to investigate the role and molecular mechanism of CMTM6 in regulating the development of thyroid cancer cells. In this study, expression levels of CMTM6 and the sodium/iodide symporter (NIS) were detected by qRT-PCR. Additionally, colony formation assay and flow cytometry were used to detect cell proliferation and apoptosis, while expression levels of various proteins were assessed using Western blotting. Further, the apoptosis and invasion capacity of cells were investigated by scratch and transwell experiments. Finally, the effect of CMTM6 on the epithelial-mesenchymal transition (EMT) of thyroid cancer cells was determined by immunofluorescence assay, which measured the expression levels of epithelial and mesenchymal phenotypic markers. The results of qRT-PCR experiments showed that CMTM6 was highly expressed in thyroid cancer tissues and cells. In addition, knockdown of CMTM6 expression significantly increased NIS expression. Function experiments demonstrated that small interfering (si)-CMTM6 treatment inhibited the proliferation, migration, invasion, and EMT of thyroid cancer cells, while promoting apoptosis of FTC133 cells. Furthermore, mechanistic studies showed that mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) phosphorylation were inhibited by si-CMTM6, as demonstrated by Western blot experiments. In conclusion, our findings demonstrated the role of CMTM6 in the metastasis of thyroid cancer. Briefly, CMTM6 exerts its tumor-promoting effect through the MAPK signaling pathway and could potentially be used as a valuable biomarker for thyroid cancer diagnosis and prognosis.

Exercise influence on monocarboxylate transporter 1 (MCT1) and 4 (MCT4) in the skeletal muscle: A systematic review.

This review aims to systematically analyze the effect of exercise on muscle MCT protein levels and mRNA expression of their respective genes, considering exercise intensity, and duration (single-exercise session and training program) in humans and rodents, to observe whether both models offer aligned results. The review also aims to report methodological aspects that need to be improved in future studies. A systematic search was conducted in the PubMed and Web of Science databases, and the Preferred Reporting Items for Systematic review and Meta-Analyses (PRISMA) checklist was followed. After applying inclusion and exclusion criteria, 41 studies were included and evaluated using the Cochrane collaboration tool for risk of bias assessment. The main findings indicate that exercise is a powerful stimulus to increase MCT1 protein content in human muscle. MCT4 protein level increases can also be observed after a training program, although its responsiveness is lower compared to MCT1. Both transporters seem to change independently of exercise intensity, but the responses that occur with each intensity and each duration need to be better defined. The effect of exercise on muscle mRNA results is less defined, and more research is needed especially in humans. Moreover, results in rodents only agree with human results on the effect of a training program on MCT1 protein levels, indicating increases in both. Finally, we addressed important and feasible methodological aspects to improve the design of future studies.

Role of sodium taurocholate cotransporting polypeptide (NTCP) in HBV-induced hepatitis: Opportunities for developing novel therapeutics.

Hepatitis B is an infectious disease caused by the HBV virus. It presents a significant challenge for treatment due to its chronic nature and the potential for developing severe complications, including hepatocirrhosis and hepatocellular carcinoma. These complications not only cause physical and psychological distress to patients but also impose substantial economic and social burdens on both individuals and society as a whole. The internalization of HBV relies on endocytosis and necessitates the involvement of various proteins, including heparin sulfate proteoglycans, epidermal growth factor receptors, and NTCP. Among these proteins, NTCP is pivotal in HBV internalization and is primarily located in the liver's basement membrane. As a transporter of bile acids, NTCP also serves as a receptor facilitating HBV entry into cells. Numerous molecules have been identified to thwart HBV infection by stifling NTCP activity, although only a handful exhibit low IC50 values. In this systematic review, our primary focus dwells on the structure and regulation of NTCP, as well as the mechanism involved in HBV internalization. We underscore recent drug breakthroughs that specifically target NTCP to combat HBV infection. By shedding light on these advances, this review contributes novel insights into developing effective anti-HBV medications.

Sodium-glucose co-transporter 2 inhibitor canagliflozin modulates myocardial metabolism and inflammation in a swine model for chronic myocardial ischemia.

BACKGROUND: Inflammation and disruption of cardiac metabolism are prevalent in the setting of myocardial ischemia. Canagliflozin, a sodium-glucose costransporter-2 inhibitor, has beneficial effects on the heart, though the precise mechanisms are unknown. This study investigated the effects of canagliflozin therapy on metabolic pathways and inflammation in ischemic myocardial tissue using a swine model of chronic myocardial ischemia. METHODS: Sixteen Yorkshire swine underwent placement of an ameroid constrictor to the left circumflex artery to induce chronic ischemia. Two weeks later, pigs received either no drug (n = 8) or 300 mg canagliflozin (n = 8) daily. Five weeks later, pigs underwent terminal harvest and tissue collection. RESULTS: Canagliflozin treatment was associated with a trend toward decreased expression of fatty acid oxidation inhibitor acetyl-CoA carboxylase and decreased phosphorylated/inactivated acetyl-CoA carboxylase, a promotor of fatty acid oxidation, compared with control ischemic myocardium (P = .08, P = .03). There was also a significant modulation in insulin resistance markers p-IRS1, p-PKCα, and phosphoinositide 3-kinase in ischemic myocardium of the canagliflozin group compared with the control group (all P < .05). Canagliflozin treatment was associated with a significant increase in inflammatory markers interleukin 6, interleukin 17, interferon-gamma, and inducible nitric oxide synthase (all P < .05). There was a trend toward decreased expression of the anti-inflammatory cytokines interleukin 10 (P = .16) and interleukin 4 (P = .31) with canagliflozin treatment. CONCLUSION: The beneficial effects of canagliflozin therapy appear to be associated with inhibition of fatty acid oxidation and enhancement of insulin signaling in ischemic myocardium. Interestingly, canagliflozin appears to increase the levels of several inflammatory markers, but further studies are required to better understand how canagliflozin modulates inflammatory signaling pathways.

Novel missense variants cause intermediate phenotypes in the phenotypic spectrum of SLC5A6-related disorders.

SLC5A6 encodes the sodium-dependent multivitamin transporter, a transmembrane protein that uptakes biotin, pantothenic acid, and lipoic acid. Biallelic SLC5A6 variants cause sodium-dependent multivitamin transporter deficiency (SMVTD) and childhood-onset biotin-responsive peripheral motor neuropathy (COMNB), which both respond well to replacement therapy with the above three nutrients. SMVTD usually presents with various symptoms in multiple organs, such as gastrointestinal hemorrhage, brain atrophy, and global developmental delay, at birth or in infancy. Without nutrient replacement therapy, SMVTD can be lethal in early childhood. COMNB is clinically milder and has a later onset than SMVTD, at approximately 10 years of age. COMNB symptoms are mostly limited to peripheral motor neuropathy. Here we report three patients from one Japanese family harboring novel compound heterozygous missense variants in SLC5A6, namely NM_021095.4:c.[221C>T];[642G>C] p.[(Ser74Phe)];[(Gln214His)]. Both variants were predicted to be deleterious through multiple lines of evidence, including amino acid conservation, in silico predictions of pathogenicity, and protein structure considerations. Drosophila analysis also showed c.221C>T to be pathogenic. All three patients had congenital brain cysts on neonatal cranial imaging, but no other morphological abnormalities. They also had a mild motor developmental delay that almost completely resolved despite no treatment. In terms of severity, their phenotypes were intermediate between SMVTD and COMNB. From these findings we propose a new SLC5A6-related disorder, spontaneously remitting developmental delay with brain cysts (SRDDBC) whose phenotypic severity is between that of SMVTD and COMNB. Further clinical and genetic evidence is needed to support our suggestion.

Inhibition of the renal apical sodium dependent bile acid transporter prevents cholemic nephropathy in mice with obstructive cholestasis.

BACKGROUND & AIMS: Cholemic nephropathy (CN) is a severe complication of cholestatic liver diseases for which there is no specific treatment. We revisited its pathophysiology with the aim of identifying novel therapeutic strategies. METHODS: Cholestasis was induced by bile duct ligation (BDL) in mice. Bile flux in kidneys and livers was visualized by intravital imaging, supported by MALDI mass spectrometry imaging and liquid chromatography-tandem mass spectrometry. The effect of AS0369, a systemically bioavailable apical sodium-dependent bile acid transporter (ASBT) inhibitor, was evaluated by intravital imaging, RNA-sequencing, histological, blood, and urine analyses. Translational relevance was assessed in kidney biopsies from patients with CN, mice with a humanized bile acid (BA) spectrum, and via analysis of serum BAs and KIM-1 (kidney injury molecule 1) in patients with liver disease and hyperbilirubinemia. RESULTS: Proximal tubular epithelial cells (TECs) reabsorbed and enriched BAs, leading to oxidative stress and death of proximal TECs, casts in distal tubules and collecting ducts, peritubular capillary leakiness, and glomerular cysts. Renal ASBT inhibition by AS0369 blocked BA uptake into TECs and prevented kidney injury up to 6 weeks after BDL. Similar results were obtained in mice with humanized BA composition. In patients with advanced liver disease, serum BAs were the main determinant of KIM-1 levels. ASBT expression in TECs was preserved in biopsies from patients with CN, further highlighting the translational potential of targeting ASBT to treat CN. CONCLUSIONS: BA enrichment in proximal TECs followed by oxidative stress and cell death is a key early event in CN. Inhibiting renal ASBT and consequently BA enrichment in TECs prevents CN and systemically decreases BA concentrations. IMPACT AND IMPLICATIONS: Cholemic nephropathy (CN) is a severe complication of cholestasis and an unmet clinical need. We demonstrate that CN is triggered by the renal accumulation of bile acids (BAs) that are considerably increased in the systemic blood. Specifically, the proximal tubular epithelial cells of the kidney take up BAs via the apical sodium-dependent bile acid transporter (ASBT). We developed a therapeutic compound that blocks ASBT in the kidneys, prevents BA overload in tubular epithelial cells, and almost completely abolished all disease hallmarks in a CN mouse model. Renal ASBT inhibition represents a potential therapeutic strategy for patients with CN.

Neuronal K+-Cl- cotransporter KCC2 as a promising drug target for epilepsy treatment.

Epilepsy is a prevalent neurological disorder characterized by unprovoked seizures. γ-Aminobutyric acid (GABA) serves as the primary fast inhibitory neurotransmitter in the brain, and GABA binding to the GABAA receptor (GABAAR) regulates Cl- and bicarbonate (HCO3-) influx or efflux through the channel pore, leading to GABAergic inhibition or excitation, respectively. The neuron-specific K+-Cl- cotransporter 2 (KCC2) is essential for maintaining a low intracellular Cl- concentration, ensuring GABAAR-mediated inhibition. Impaired KCC2 function results in GABAergic excitation associated with epileptic activity. Loss-of-function mutations and altered expression of KCC2 lead to elevated [Cl-]i and compromised synaptic inhibition, contributing to epilepsy pathogenesis in human patients. KCC2 antagonism studies demonstrate the necessity of limiting neuronal hyperexcitability within the brain, as reduced KCC2 functioning leads to seizure activity. Strategies focusing on direct (enhancing KCC2 activation) and indirect KCC2 modulation (altering KCC2 phosphorylation and transcription) have proven effective in attenuating seizure severity and exhibiting anti-convulsant properties. These findings highlight KCC2 as a promising therapeutic target for treating epilepsy. Recent advances in understanding KCC2 regulatory mechanisms, particularly via signaling pathways such as WNK, PKC, BDNF, and its receptor TrkB, have led to the discovery of novel small molecules that modulate KCC2. Inhibiting WNK kinase or utilizing newly discovered KCC2 agonists has demonstrated KCC2 activation and seizure attenuation in animal models. This review discusses the role of KCC2 in epilepsy and evaluates its potential as a drug target for epilepsy treatment by exploring various strategies to regulate KCC2 activity.

OCTN1 (SLC22A4) displays two different transport pathways for organic cations or zwitterions.

BACKGROUND: OCTN1 belongs to the SLC22 family, which includes transporters for cationic, zwitterionic, and anionic substrates. OCTN1 function and role in cells are still poorly understood. Not only cations, such as TEA, but also zwitterions, such as carnitine and ergothioneine, figure among transported molecules. METHODS: In this work, we carried out transport assays measuring [14C]-TEA and [3H]-Carnitine in proteoliposomes reconstituted with the recombinant human OCTN1 in the presence of Na+ or other cations. The homology model of OCTN1 was built using the structure of OCT3 as a template for docking analysis. RESULTS: TEA and carnitine did not inhibit each other. Moreover, carnitine uptake was not affected by the presence of Na+ and TEBA, whereas TEA was strongly inhibited by both compounds. Computational data revealed that TEA, Na+, and carnitine can interact with E381 in the OCTN1 substrate site. Differently from TEA, in the presence of Na+, carnitine is still able to interact with the binding site via R469. CONCLUSIONS: The lack of mutual inhibition of the two prototype substrates, the different effect of Na+ and TEBA on their transport reaction, together with the computational analysis supports the existence of two transport pathways for cations and zwitterions. GENERAL SIGNIFICANCE: The results shed new light on the transport mechanisms of OCTN1, helping to get further insights into the structure/function relationships. The described results correlate well with previous and very recent findings on the polyspecificity of the OCT group of transporters belonging to the same family.