Laevis Oocyte Expression System Research Articles

Amantadine inhibits known and novel ion channels encoded by SARS-CoV-2 in vitro

The dire need for COVID-19 treatments has inspired strategies of repurposing approved drugs. Amantadine has been suggested as a candidate, and cellular as well as clinical studies have indicated beneficial effects of this drug. We demonstrate that amantadine and hexamethylene-amiloride (HMA), but not rimantadine, block the ion channel activity of Protein E from SARS-CoV-2, a conserved viroporin among coronaviruses. These findings agree with their binding to Protein E as evaluated by solution NMR and molecular dynamics simulations. Moreover, we identify two novel viroporins of SARS-CoV-2; ORF7b and ORF10, by showing ion channel activity in a X. laevis oocyte expression system. Notably, amantadine also blocks the ion channel activity of ORF10, thereby providing two ion channel targets in SARS-CoV-2 for amantadine treatment in COVID-19 patients. A screen of known viroporin inhibitors on Protein E, ORF7b, ORF10 and Protein 3a from SARS-CoV-2 revealed inhibition of Protein E and ORF7b by emodin and xanthene, the latter also blocking Protein 3a. This illustrates a general potential of well-known ion channel blockers against SARS-CoV-2 and specifically a dual molecular basis for the promising effects of amantadine in COVID-19 treatment.

Kv3.1 channelopathy: a novel loss-of-function variant and the mechanistic basis of its clinical phenotypes.

BackgroundKCNC1 encodes Kv3.1, a subunit of the Kv3 voltage-gated potassium channels. It is predominantly expressed in inhibitory GABAergic interneurons and cerebellar neurons. Kv3.1 channelopathy has been linked to a variety of human diseases including epilepsy, developmental delay, and ataxia. Characterization of structural and functional disturbances of this channel, and its relationship to a heterogenous group of clinical phenotypes, is a current topic of research. We herein characterize the clinical phenotype as well as the functional and structural consequences of the novel KCNC1 p.R317S variant. We further set out to explore the mechanistic basis for the spectrum of KCNC1 related channelopathies.MethodsVariant was identified via whole-exome sequencing and its functional impact was determined using two-electrode voltage clamp recordings in Xenopus laevis oocytes. Homolog modeling and in silico structural analysis were performed on the p.R317S variant and other KCNC1 related variants.ResultsWe identified a novel loss-of-function KCNC1 variant c.949C>A (p.R317S) presenting with symptoms similar to myoclonic epilepsy and ataxia due to potassium channel (MEAK), but with distinct radiological features. Functional analysis in the Xenopus laevis oocyte’s expression system revealed that the current amplitudes were significantly decreased in the p.R317S variant compared to the wild type, indicating a dominant-negative effect. Atomic structural analysis of the KCNC1 related variants provided a possible mechanistic explanation for the heterogeneity in the clinical spectrum.ConclusionsWe have identified the p.R317S loss-of-function variant in the KCNC1 gene, expanded the spectrum of potassium channelopathy and provided mechanistic insights into KCNC1 related disorders.

Transport of 2,4-dichloro phenoxyacetic acid by human Na+-coupled monocarboxylate transporter 1 (hSMCT1, SLC5A8)

Using X. laevis oocyte expression system, we investigated whether human Na+-coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na+-dependent and electrogenic. The Na+-dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na+-dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na+-dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity.

Structure-activity relationships of bumetanide derivatives: correlation between diuretic activity in dogs and inhibition of the human NKCC2A transporter.

The N-K-Cl cotransporters (NKCCs) mediate the coupled, electroneutral movement of Na+ , K+ and Cl- ions across cell membranes. There are two isoforms of this cation co-transporter, NKCC1 and NKCC2. NKCC2 is expressed primarily in the kidney and is the target of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure-activity studies on effects of bumetanide derivatives on NKCC2 are not available. In this study, the effect of a series of diuretically active bumetanide derivatives was investigated on human NKCC2 variant A (hNKCC2A) expressed in Xenopus laevis oocytes. Bumetanide blocked hNKCC2A transport with an IC50 of 4 μM. There was good correlation between the diuretic potency of bumetanide and its derivatives in dogs and their inhibition of hNKCC2A (r2 = 0.817; P < 0.01). Replacement of the carboxylic group of bumetanide by a non-ionic residue, for example, an anilinomethyl group, decreased inhibition of hNKCC2A, indicating that an acidic group was required for transporter inhibition. Exchange of the phenoxy group of bumetanide for a 4-chloroanilino group or the sulfamoyl group by a methylsulfonyl group resulted in compounds with higher potency to inhibit hNKCC2A than bumetanide. The X. laevis oocyte expression system used in these experiments allowed analysis of the structural requirements that determine relative potency of loop diuretics on human NKCC2 splice variants, and may lead to the discovery of novel high-ceiling diuretics.

Placental uric acid transporter GLUT9 is modulated by free iodine

PLACENTAL URIC ACID TRANSPORTER GLUT9 IS MODULATED BY FREE IODINE Objectives: Materno-fetal transplacental transport is crucial for the fetal well-being. The altered expression of placental transport proteins under specific pathophysiological conditions may affect the intrauterine environment. Pre-eclampsia is often associated with high maternal uric acid serum levels. The regulation of the placental uric transport system and its transporter glucose transporter (GLUT)-9 are not fully understood yet. The aim of this study was to investigate the placental urate transport and to characterize its transporter GLUT9. Methods: In this study we used a transepithelial transport (Transwell®) model to assess uric acid transport activity. Electrophysiological techniques and radioactive ligand up-take assays were used to measure transport activity of GLUT9 expressed in Xenopus oocytes. Results: In the Transwell/model uric acid is transported across the BeWo choriocarcinoma cell monolayer with 530 pmol/min at the linear stage. We could successfully over-express GLUT9 using the Xenopus laevis oocytes expression system. Chloride modulates the urate transport system: interestingly replacing chloride with iodine resulted in a complete loss of urate transport activity.We determined the IC50 of iodine at 30uM concentration. In radioactive up-take experiments iodinehad noeffect on uric acid transport. Conclusions: In vitro the “materno-fetal” transport of uric acid is slow. This indicates that in vivo the child is protected from short-term fluctuations of maternal uric acid serum concentrations. The different results regarding iodine-mediated regulation of GLUT9 transport activity between electrophysiological and radioactive ligand uptake experiments may suggest that iodine does not directly inhibit uric acid transport, but changes the mode of up-take from an electrogenic to an electroneutral transport. GLUT9 is not an uric acid uniporter, there are more ions involved in the transport. This may allow regulating uric acid transport by the change from an active to a passive transport.

Functional and electrophysiological characterization of four non‐truncating mutations responsible for creatine transporter (SLC6A8) deficiency syndrome

Intellectual disability coupled with epilepsy are clinical hallmarks of the creatine (Cr) transporter deficiency syndrome resulting from mutations in the SLC6A8 gene. So far characterization of pathogenic mutations of SLC6A8 has been limited to Cr uptake. The aim of our study was to characterize the electrogenic and pharmacological properties of non truncating SLC6A8 mutations identified in patients presenting variable clinical severity. Electrophysiological and pharmacological properties of four mutants (including two novel ones) were studied in X. laevis oocyte expression system. Creatine uptake was assessed with [(14)C]-Cr in X. laevis and patients' fibroblasts. Subcellular localization was determined by immunofluorescence and western blot. All mutants were properly targeted to the plasma membrane in both systems. Mutations led to the complete loss of both electrogenic and transport activities in X. laevis and Cr uptake in patients' fibroblasts. Among the Cr analogs tested, guanidinopropionate induced an electrogenic activity with the normal SLC6A8 transporter similar to creatine whereas a phosphocreatine derivative, PCr-Mg-CPLX, resulted in partial activity. SLC6A8 mutants displayed no electrogenic activity with all Cr analogs tested in X. laevis oocytes. Although the mutations altered various domains of SLC6A8 Cr uptake and electrogenic properties were completely inhibited and could not be dissociated. Besides the metabolic functions of Cr, the loss of SLC6A8 electrogenic activity, demonstrated here for the first time, may also play a role in the altered brain functions of the patients.

Abstract 4459: a-Methyltryptophan (a-MT) inhibits estrogen receptor (ER)-positive breast cancer growth through blockade of SLC6A14

Abstract Introduction: SLC6A14 is an amino acid transporter that transports all essential amino acids as well as glutamine and arginine, and is energized by Na+ gradient, Cl- gradient, and membrane potential. The expression of SLC6A14 is upregulated in ER-positive breast cancer cells but not in ER-negative breast cancer cells. Since cancer cells have an increased need for essential amino acids (protein synthesis), Gln (nucleotide synthesis/glutaminolysis), and Arg (essential amino acid in cancer), we hypothesize that cancer cells satisfy their need for these amino acids by upregulating SLC6A14, and that blockade of SLC6A14 offers a rational strategy for treatment of ER-positive breast cancer. Methods: The transport of Leu, Gln, and Arg via SLC6A14 was studied in X. laevis oocyte expression system. SLC6A14 expression was monitored by RT-PCR and immunohistochemistry in normal and tumors (ER-positive and ER-negative) from primary breast cancer specimens. The influence of estrogen on SLC6A14 expression was investigated in ER-positive BT474 breast cancer cells. α-MT was used as the blocker of SLC6A14. The differential effects of α-MT on MCF7 (ER-positive), MB231 (ER-negative) and MCF10A (non-malignant) cells were investigated in terms of mTOR activity, autophagy, and apoptosis. The ability of α-MT to inhibit the growth of breast cancer cells was evaluated by xenograft in nude mice. α-MT was administered to mice in drinking water (2 mg/ml). Plasma levels of α-MT were measured by HPLC. Results: The transport of Leu, Gln, and Arg via SLC6A14 was dependent on Na+ and Cl- with a Na+:Cl-:amino acid stoichiometry of 2:1:1, and was blocked by α-MT. SLC6A14 expression was upregulated in ER-positive breast cancer. SLC6A14 was not expressed in ER-negative breast cancer. Estrogen induced SLC6A14 expression in ER-positive breast cancer cells. Exposure of ER-positive MCF7 breast cancer cells to α-MT induced the expression of asparagine synthetase and CHOP, inhibited mTOR activity, and induced autophagy and apoptosis. When combined with an autophagy inhibitor, α-MT induced apoptosis in MCF7 cells more potently. These effects were specific for ER-positive breast cancer cells, and were not seen in ER-negative cells or in non-malignant cells. In mouse xenografts, α-MT reduced the growth of ER-positive ZR-75-1 breast cancer cells, but did not have any effect on the growth of ER-negative MB231 cells. MCF7 cells did not grow in nude mice unless estrogen was administered, and under these conditions, α-MT did not block the growth of the cells. Plasma concentration of α-MT in treated mice was 8.5 ± 1.5 μM. α-MT was not detectable in the plasma of untreated mice. Conclusions: SLC6A14 is an ideal and effective drug target for treatment of ER-positive breast cancer. α-MT has potential for use as an anticancer drug, and may also serve as a lead compound in the design/development of newer blockers of SLC6A14 for use in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2011-4459

Abstract 2198: Role of SLC5A8, a plasma membrane transporter and a tumor suppressor, in the antitumor activity of dichloroacetate

Abstract Introduction: There has been growing interest among the public and scientists in dichloroacetate as a potential anticancer drug. Credible evidence exists for the antitumor activity of this compound, but high concentrations are needed for significant therapeutic effect. Unfortunately, these high concentrations produce detrimental side effects involving nervous system, hence precluding its use for cancer treatment. The mechanistic basis of the compound's antitumor activity is its ability to activate pyruvate dehydrogenase complex through inhibition of pyruvate dehydrogenase kinase. Since the compound inhibits the kinase at micromolar concentrations, it is not known why therapeutically prohibitive high doses are needed for suppression of tumor growth. We hypothesized that lack of effective mechanisms for the entry of dichloroacetate into tumor cells may underlie this phenomenon. Methods: The transport of acetate and its chloro derivatives by human SLC5A8 was studied using the X. laevis oocytes expression system. The human transporter was expressed in oocytes heterologously by injection of SLC5A8 cRNA. The transport function was monitored electrophysiologically by the two-microelectrode voltage-clamp technique. Breast, colon and prostate cancer cell lines were transfected with SLC5A8 and expression was monitored by RT-PCR analysis. Intracellular levels of pyruvate and HDAC activity was also monitored in these cell lines. Results: SLC5A8 transports dichloroacetate very effectively and with high affinity. This transporter is expressed in normal cells, but the expression is silenced in tumor cells via epigenetic mechanisms. The lack of the transporter makes tumor cells resistant to the antitumor activity of dichloroacetate. However, if the transporter is expressed in tumor cells ectopically, the cells become sensitive to the drug at low concentrations. This is evident in breast cancer cells, colon cancer cells, and prostate cancer cells. Normal cells, which constitutively express the transporter, are however not affected by the compound, indicating the tumor cell-selective therapeutic activity. The mechanism of the antitumor activity of the compound depends on its ability to inhibit pyruvate dehydrogenase kinase and force mitochondrial oxidation of pyruvate. Conclusions: The silencing of SLC5A8 in tumors involves DNA methylation and its expression can be induced by treatment with DNA methylation inhibitors. Our findings suggest that combining dichloroacetate with a DNA methylation inhibitor would offer a means to reduce the doses of dichloroacetate to avoid detrimental effects associated with high doses but without compromising antitumor activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2198. doi:10.1158/1538-7445.AM2011-2198