Xenopus Laevis Oocyte Expression System Research Articles

Expression of the grapevine anion transporter ALMT2 in Arabidopsis root decreases shoot Cl-/NO3- ratio under salt stress.

Grapevines (Vitis vinifera, Vvi) are economically important crop plants which, when challenged with salt (NaCl) in soil and/or irrigation water, tend to accumulate Na+ and Cl- in aerial tissues impacting yield, and berry acceptability for winemaking. Grapevine (Vitis spp.) rootstocks vary in their capacity for shoot Cl- exclusion. Here, we characterise two putative anion transporter genes - Aluminium-activated Malate Transporter VviALMT2 and VviALMT8 - that were differentially expressed in the roots of efficient (140 Ruggeri) and inefficient (K51-40) Cl- excluding rootstocks, to explore their potential for impacting shoot Cl- exclusion. Using the Xenopus laevis oocyte expression system, VviALMT2 and VviALMT8 formed conductive channels that were highly permeable to NO3-, slightly-to-moderately permeable to other substrates including Cl- and malate, but impermeable to SO42-. RT-qPCR analyses revealed that VviALMT2 was more highly expressed in the root vasculature and up-regulated by high [NO3-] re-supply post starvation, while fluorescently tagged translational fusion VviALMT2 localised to the plasma membrane. As VviALMT8 showed no such features, we selected VviALMT2 as our salt exclusion candidate and assessed its function in planta. Expression of VviALMT2 in Arabidopsis thaliana root vasculature reduced shoot [Cl-]/[NO3-] after NaCl treatment, which suggests that VviALMT2 can be beneficial to plants under salt stress.

Characterization of a vacuolar importer of secologanin in Catharanthus roseus

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. Biochemical characterization was conducted using the Xenopus laevis oocyte expression system to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, translocating 1 mM of substrate within 25 min. The transporter displayed strict directionality and specificity for secologanin and did not accept other secoiridoid substrates. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of pathway flux, mediating the balance between a defense arsenal and cellular homeostasis.

Functional characterization of Clonorchis sinensis choline transporter.

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 μM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.

A Simplified Protocol to Incorporate the Fluorescent Unnatural Amino Acid ANAP into Xenopus laevis Oocyte-Expressed P2X7 Receptors.

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein.

Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

PurposesThis study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict the three-dimensional structure change of human Na–Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo.MethodsSLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, China National Knowledge Infrastructure, and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R, and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevisoocyte expression system. In the study, 35 GS patients and 20 healthy volunteers underwent the thiazide test.ResultsT60M, T163M, D486N, R913Q, R928C, and R959frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all six mutations: T60M 22 ± 9.2%, R928C 29 ± 12%, L215F 38 ± 14%, N534K 41 ± 15.5%, Q617R 63 ± 22.1%, and D486N 77 ± 20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations.ConclusionsFrequent mutations (T60M, D486N, and R928C) and novel mutations (L215F, N534K, and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on the patients.

Functional characterization of Clonorchis sinensis sodium-bile acid co-transporter (CsSBAT) as a steroid sulfate transporter.

Clonorchis sinensis (Cs) is a common trematode in Asian countries. Infection by Cs can result in many clinical symptoms. Here, a cDNA encoding a Cs apical sodium-dependent bile acid transporter (CsSBAT) was isolated from a Cs cDNA library, and functional characterization was performed using Xenopus laevis oocyte expression system. When expressed in Xenopus laevis oocytes, CsSBAT mediated the transport of radiolabeled estrone sulfate and dehydroepiandrosterone sulfate. No trans-uptake of carnitine, estradiol 17 β-D glucuronide, prostaglandin E2, p-aminohippuric acid, α-ketoglutaric acid, and tetraethylammonium was observed. CsSBAT-mediated estrone sulfate uptake was in a time- and sodium-dependent manner. CsSBAT showed no exchange properties in efflux experiments. Concentration-dependent results showed saturable kinetics consistent with the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 0.3 ± 0.04μM for [3H]estrone sulfate. CsSBAT-mediated estrone sulfate uptake was strongly inhibited by sulfate conjugates but not glucuronide conjugates. These findings contribute to our understanding of CsSBAT transport properties and the cascade of estrogen metabolite movement in Cs.

WIN55,212-2, a Dual Modulator of Cannabinoid Receptors and G Protein-Coupled Inward Rectifier Potassium Channels.

The coupling of cannabinoid receptors, CB1 and CB2, to G protein-coupled inward rectifier potassium channels, GIRK1 and GIRK2, modulates neuronal excitability in the human brain. The present study established and validated the functional expression in a Xenopus laevis oocyte expression system of CB1 and CB2 receptors, interacting with heteromeric GIRK1/2 channels and a regulator of G protein signaling, RGS4. This ex vivo system enables the discovery of a wide range of ligands interacting orthosterically or allosterically with CB1 and/or CB2 receptors. WIN55,212-2, a non-selective agonist of CB1 and CB2, was used to explore the CB1- or CB2-GIRK1/2-RGS4 signaling cascade. We show that WIN55,212-2 activates CB1 and CB2 at low concentrations whereas at higher concentrations it exerts a direct block of GIRK1/2. This illustrates a dual modulatory function, a feature not described before, which helps to explain the adverse effects induced by WIN55,212-2 in vivo. When comparing the effects with other typical cannabinoids such as Δ9-THC, CBD, CP55,940, and rimonabant, only WIN55,212-2 can significantly block GIRK1/2. Interestingly, the inward rectifier potassium channel, IRK1, a non-G protein-coupled potassium channel important for setting the resting membrane voltage and highly similar to GIRK1 and GIRK2, is not sensitive to WIN55,212-2, Δ9-THC, CBD, CP55,940, or rimonabant. From this, it is concluded that WIN55,212-2 selectively blocks GIRK1/2.

Zymogen-locked mutant prostasin (Prss8) leads to incomplete proteolytic activation of the epithelial sodium channel (ENaC) and severely compromises triamterene tolerance in mice.

The serine protease prostasin (Prss8) is expressed in the distal tubule and stimulates proteolytic activation of the epithelial sodium channel (ENaC) in co-expression experiments in vitro. The aim of this study was to explore the role of prostasin in proteolytic ENaC activation in the kidney in vivo. We used genetically modified knockin mice carrying a Prss8 mutation abolishing proteolytic activity (Prss8-S238A) or a mutation leading to a zymogen-locked state (Prss8-R44Q). Mice were challenged with low sodium diet and diuretics. Regulation of ENaC activity by Prss8-S238A and Prss8-R44Q was studied in vitro using the Xenopus laevis oocyte expression system. Co-expression of murine ENaC with Prss8-wt or Prss8-S238A in oocytes caused maximal proteolytic ENaC activation, whereas ENaC was activated only partially in oocytes co-expressing Prss8-R44Q. This was paralleled by a reduced proteolytic activity at the cell surface of Prss8-R44Q expressing oocytes. Sodium conservation under low sodium diet was preserved in Prss8-S238A and Prss8-R44Q mice but with higher plasma aldosterone concentrations in Prss8-R44Q mice. Treatment with the ENaC inhibitor triamterene over four days was tolerated in Prss8-wt and Prss8-S238A mice, whereas Prss8-R44Q mice developed salt wasting and severe weight loss associated with hyperkalemia and acidosis consistent with impaired ENaC function and renal failure. Unlike proteolytically inactive Prss8-S238A, zymogen-locked Prss8-R44Q produces incomplete proteolytic ENaC activation in vitro and causes a severe renal phenotype in mice treated with the ENaC inhibitor triamterene. This indicates that Prss8 plays a role in proteolytic ENaC activation and renal function independent of its proteolytic activity.

Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis

Mice lacking connexin 30 (Cx30) display increased epithelial sodium channel (ENaC) activity in the distal nephron and develop salt-sensitive hypertension. This indicates a functional link between Cx30 and ENaC, which remains incompletely understood. Here, we explore the effect of Cx30 on ENaC function using the Xenopus laevis oocyte expression system. Coexpression of human Cx30 with human αβγENaC significantly reduced ENaC-mediated whole-cell currents. The size of the inhibitory effect on ENaC depended on the expression level of Cx30 and required Cx30 ion channel activity. ENaC inhibition by Cx30 was mainly due to reduced cell surface ENaC expression resulting from enhanced ENaC retrieval without discernible effects on proteolytic channel activation and single-channel properties. ENaC retrieval from the cell surface involves the interaction of the ubiquitin ligase Nedd4-2 with PPPxY-motifs in the C-termini of ENaC. Truncating the C- termini of β- or γENaC significantly reduced the inhibitory effect of Cx30 on ENaC. In contrast, mutating the prolines belonging to the PPPxY-motif in γENaC or coexpressing a dominant-negative Xenopus Nedd4 (xNedd4-CS) did not significantly alter ENaC inhibition by Cx30. Importantly, the inhibitory effect of Cx30 on ENaC was significantly reduced by Pitstop-2, an inhibitor of clathrin-mediated endocytosis, or by mutating putative clathrin adaptor protein 2 (AP-2) recognition motifs (YxxФ) in the C termini of β- or γ-ENaC. In conclusion, our findings suggest that Cx30 inhibits ENaC by promoting channel retrieval from the plasma membrane via clathrin-dependent endocytosis. Lack of this inhibition may contribute to increased ENaC activity and salt-sensitive hypertension in mice with Cx30 deficiency.

A new system to evaluate characteristics of Niemann-Pick C1 Like 1-mediated cholesterol transport using Xenopus laevis oocytes

Niemann-Pick C1 Like 1 (NPC1L1) is known to be involved in the intestinal absorption of cholesterol. For evaluating the function of NPC1L1, cell lines such as Caco-2, Madin-Darby canine kidney (MDCK) II, and McA-RH7777 have been used in previous studies, but the detailed molecular mechanism of transport has not been elucidated. In this study, the characteristics of cholesterol transport via NPC1L1 were investigated using a Xenopus laevis oocyte expression system in addition to a conventional cell line with stable expression. The transport activity of cholesterol uptake was increased in NPC1L1-overexpressed MDCK cells compared with that in mock cells, but MDCK cells expressed endogenous NPC1L1 and had high cholesterol transport activity. On the other hand, cRNA-injected oocytes expressed NPC1L1 after culturing for 5–6 days. The transport activity of cholesterol uptake was increased in NPC1L1 cRNA-injected oocytes compared with that in water-injected oocytes. In addition, the uptake of cholesterol was decreased in the presence of ezetimibe, an NPC1L1 inhibitor, in cRNA-injected oocytes but not in control oocytes, indicating that endogenous NPC1L1 is not expressed in oocytes. Furthermore, cholesterol uptake was substantially decreased in NPC1L1 L216A cRNA-injected oocytes compared with that in NPC1L1 cRNA-injected oocytes, indicating that leucine at position 216 of NPC1L1 is important for cholesterol transport and that an oocyte expression system is useful for mutant analysis. These results indicate that the oocyte expression system is useful for evaluating the characteristics of NPC1L1-mediated cholesterol transport and may contribute to the elucidation of the detailed molecular mechanism of cholesterol transport via NPC1L1.

Three Host Plant Volatiles, Hexanal, Lauric Acid, and Tetradecane, are Detected by an Antenna-Biased Expressed Odorant Receptor 27 in the Dark Black Chafer Holotrichia parallela.

Insects rely on olfaction to locate their host plants by antennae in complex chemical environments. Odorant receptor (OR) genes are thought to play a crucial role in the process. ORs function together with odorant coreceptors to determine the specificity and sensitivity of olfactory reception. The dark black chafer, Holotrichia parallela Motschulsky (Coleoptera: Scarabaeidae), is a destructive underground pest. To understand the molecular basis of H. parallela olfactory reception, an olfactory-biased expressed odorant receptor HparOR27 and HparOrco (HparOR40) were identified from antennal transcriptome analysis and prediction of the sequence structure. Tissue expression analysis showed that HparOR27 was mainly expressed in adult antennae throughout developmental stages. The functions of HparOR27 were analyzed using the Xenopus laevis oocyte expression system. HparOR27 was broadly responsive to three host plant volatiles, including hexanal, lauric acid, and tetradecane. Electroantennogram tests confirmed that three ligands were electrophysiologically active in antennae of female adults. A Y-tube olfactometer test indicated that hexanal was a repellent for adults of both sexes. Taken together, our data support the identification of odorant receptors and provide a molecular basis for eco-friendly pest control.

Extracellular lysine 38 plays a crucial role in pH-dependent transport via human monocarboxylate transporter 1

Human monocarboxylate transporters (hMCTs) are expressed in many tissues and mediate the transport of various substrates across the plasma membrane. Among hMCTs, hMCT1–4 cotransport H+ with monocarboxylates such as pyruvate and l-lactate, implying that these proteins recognize both substrate and H+. However, the mechanism of translocation, and particularly that of hMCT1 pH-dependent transport, remains largely unknown. This study aimed at identifying residues involved in the pH dependence of hMCT1 using a combination of amino acid-modifying reagents, site-directed mutagenesis in a Xenopus laevis oocyte expression system, and homology modeling. We showed that diethyl pyrocarbonate (DEPC), phenylglyoxal (PGO), and 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS), which react with histidine, arginine, and lysine residues respectively, all inhibited hMCT1 activity. Since DEPC, PGO, and DIDS are membrane impermeable reagents, we mutated to other residues individual histidine, arginine, and lysine residues located within the extracellular regions of hMCT1. Analyses of these mutants demonstrated that except for K38, the extracellular basic residues of hMCT1 were not involved in its transport activity and pH dependence. Moreover, analyses of various mutants in which K38 was substituted for another residue and of an hMCT1 homology model focusing on the location of K38 in the three-dimensional structure delineated the mechanism of hMCT1 pH dependence. Collectively, our data indicate that K38 plays an essential role in hMCT1 transport activity. We would like to propose a mechanism whereby K38 is positioned within a hydrophobic and narrow cavity that is part of the transport pathway, and regulates pH-dependent gating of hMCT1.

Stereoselective Anti-Cancer Activities of Ginsenoside Rg3 on Triple Negative Breast Cancer Cell Models.

Ginsenoside Rg3 (Rg3) has two epimers, 20(S)-ginsenoside Rg3 (SRg3) and 20(R)-ginsenoside Rg3 (RRg3), and while Rg3 itself has been reported to have anti-cancer properties, few studies have been reported on the anti-cancer effects of the different epimers. The aim was to investigate the stereoselective effects of the Rg3 epimers on triple negative breast cancer (TNBC) cell lines, tested using cell-based assays for proliferation, apoptosis, cell cycle arrest, migration and invasion. Molecular docking showed that Rg3 interacted with the aquaporin 1 (AQP1) water channel (binding score −9.4 kJ mol−1). The Xenopus laevis oocyte expression system was used to study the effect of Rg3 epimers on the AQP1 water permeability. The AQP1 expression in TNBC cell lines was compared with quantitative-polymerase chain reaction (PCR). The results showed that only SRg3 inhibited the AQP1 water flux and inhibited the proliferation of MDA-MB-231 (100 μM), due to cell cycle arrest at G0/G1. SRg3 inhibited the chemoattractant-induced migration of MDA-MB-231. The AQP1 expression in MDA-MB-231 was higher than in HCC1143 or DU4475 cell lines. These results suggest a role for AQP1 in the proliferation and chemoattractant-induced migration of this cell line. Compared to SRg3, RRg3 had more potency and efficacy, inhibiting the migration and invasion of MDA-MB-231. Rg3 has stereoselective anti-cancer effects in the AQP1 high-expressing cell line MDA-MB-231.

Encephalopathies with KCNC1 variants: genotype-phenotype-functional correlations.

ObjectiveTo analyze clinical phenotypes associated with KCNC1 variants other than the Progressive Myoclonus Epilepsy‐causing variant p.Arg320His, determine the electrophysiological functional impact of identified variants and explore genotype‐phenotype‐physiological correlations.MethodsTen cases with putative pathogenic variants in KCNC1 were studied. Variants had been identified via whole‐exome sequencing or gene panel testing. Clinical phenotypic data were analyzed. To determine functional impact of variants detected in the Kv3.1 channel encoded by KCNC1, Xenopus laevis oocyte expression system and automated two‐electrode voltage clamping were used.ResultsSix unrelated patients had a Developmental and Epileptic Encephalopathy and a recurrent de novo variant p.Ala421Val (c.1262C > T). Functional analysis of p.Ala421Val revealed loss of function through a significant reduction in whole‐cell current, but no dominant‐negative effect. Three patients had a contrasting phenotype of Developmental Encephalopathy without seizures and different KCNC1 variants, all of which caused loss of function with reduced whole‐cell currents. Evaluation of the variant p.Ala513Val (c.1538C > T) in the tenth case, suggested it was a variant of uncertain significance.InterpretationThese are the first reported cases of Developmental and Epileptic Encephalopathy due to KCNC1 mutation. The spectrum of phenotypes associated with KCNC1 is now broadened to include not only a Progressive Myoclonus Epilepsy, but an infantile onset Developmental and Epileptic Encephalopathy, as well as Developmental Encephalopathy without seizures. Loss of function is a key feature, but definitive electrophysiological separation of these phenotypes has not yet emerged.

Regulation of Na+-K+-Cl- cotransporter type 2 by the with no lysine kinase-dependent signaling pathway.

Na+-K+-Cl- cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl-] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the Xenopus laevis oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation.

Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

Claudins (cldns) represent the largest family of transmembrane tight junction (TJ) proteins, determining organ-specific epithelial barrier properties. Because methods for the analysis of multiple cldn interaction are limited, we have established the heterologous Xenopus laevis oocyte expression system for TJ protein assembly and interaction analysis. Oocytes were injected with cRNA encoding human cldn-1, -2, or -3 or with a combination of these and were incubated in pairs for interaction analysis. Immunoblotting and immunohistochemistry were performed, and membrane contact areas were analyzed morphometrically and by freeze fracture electron microscopy. Cldns were specifically detected in membranes of expressing oocytes, and coincubation of oocytes resulted in adhesive contact areas that increased with incubation time. Adjacent membrane areas revealed specific cldn signals, including "kissing-point"-like structures representing homophilic trans-interactions of cldns. Contact areas of oocytes expressing a combination markedly exceeded those expressing single cldns, indicating effects on adhesion. Ultrastructural analysis revealed a self-assembly of TJ strands and a cldn-specific strand morphology.-Vitzthum, C., Stein, L., Brunner, N., Knittel, R., Fallier-Becker, P., Amasheh, S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

An odorant receptor mediates the attractiveness of cis-jasmone to Campoletis chlorideae, the endoparasitoid of Helicoverpa armigera.

Parasitic wasps rely on olfaction to locate their hosts in complex chemical environments. Odorant receptors (ORs) function together with well-conserved odorant coreceptors (ORcos) to determine the sensitivity and specificity of olfactory reception. Campoletis chlorideae (Hymenoptera: Ichneunmonidae) is a solitary larval endoparasitoid of the cotton bollworm, Helicoverpa armigera, and some other noctuid species. To understand the molecular basis of C.chlorideae's olfactory reception, we sequenced the transcriptome of adult male and female heads (including antennae) and identified 211 OR transcripts, with 95 being putatively full length. The tissue expression profiles, as assessed by reverse-transcription PCR, showed that seven ORs were expressed only or more highly in female antennae. Their functions were analysed using the Xenopu slaevis oocyte expression system and two-electrode voltage-clamp recordings. CchlOR62 was tuned to cis-jasmone, which was attractive to female C.chlorideae adults and H.armigera larvae in the subsequent behavioural assays. Further bioassays using caged plants showed that the parasitism rate of H.armigera larvae by C.chlorideae on cis-jasmone-treated tobacco plants was higher than on the control plants. Thus, cis-jasmone appears to be an important infochemical involved in the interactions of plants, H.armigera and C.chlorideae, and CchlOR62 mediates the attractiveness of cis-jasmone to C.chlorideae.

Human Mutations in SLC2A9 (Glut9) Affect Transport Capacity for Urate.

SLC2A9 or Glut9 is a voltage sensitive urate transporter, mainly expressed in the kidneys, the liver, and the intestine. Human Glut9 loss-of-function mutations were identified in familial hypouricemia, and several single nucleotide polymorphisms (SNPs) were associated with lower serum urate, further indicating that Glut9 is a major determinant of serum uric acid level. To get insights in Glut9 transport characteristics, we systematically analyzed the function of known human Glut9 mutants using 14C-urate uptake assay and two-electrode voltage clamp (TEVC) in the Xenopus laevis oocyte expression system. Surface expression was assessed by immunostaining and biotinylation. We found decreased urate transport by flux studies for most of the variants. No variant was permissive for glucose transport. We could further differentiate two behaviors among the mutants: those harboring poor overall and cell-surface expression leading to low activity and those fully expressed at the cell surface, but presenting decreased activity. We studied the latter by TEVC and observed, in depolarized conditions, decreased inward currents measured in presence of 400 μM urate, partially reversed in 1 mM urate. In addition, we showed that C210F displays lower transport ability. By contrast, N333S showed decreased urate transport activity and urate affinity, suggesting that it may belong to the urate binding pocket. Systematic analysis of Glut9 mutants confirms Glut9 as putative target for the treatment of hyperuricemia and brings new insights in Glut9 structure – function.

DISTINCT EFFECTS OF GLUTATHIONE DISULFIDE ON δ‐ AND α‐EPITHELIAL SODIUM CHANNEL SUBUNITS

BackgroundAmiloride‐sensitive epithelial sodium channel (ENaC) is a membrane bound ion channel with selective inward permeability to Na. ENaC plays a critically important role in maintaining net fluid homeostasis. To date four ENaC subunits (α‐, β‐, γ‐, and δ‐ENaC) have been cloned in mammalian species. Mice, however, only express αβγ. In humans, an additional ENaC subunit (δ1) and its slice variant (δ2) have been recently identified. Unlike αβγ‐ENaC, the physiological role and regulation of channels made up of δ1 and δ2 subunits remain unclear.HypothesisWe tested the hypothesis that sodium channels containing α and δ ENaC subunits (alongside β and γ) are differentially regulated under oxidative stress.MethodsUsing the Xenopus laevis oocyte expression system, full‐length cDNAs for human ENaC subunits, and two electrode voltage clamp measurements, we modeled antioxidant depletion (a form of oxidative stress) and compared the effects of oxidized glutathione (GSSG) on αβγ‐ENaC and δβγ‐ENaC whole cell current. High extracellular GSSG treatments (400μM) increases the redox potential, and models antioxidant depletion in an oocyte expression system. This experimental approach allows for the singular study of αβγ vs. δβγ ENaC in a cell model system capable of normal assembly of multimeric ion channels into the membrane.ResultsGSSG significantly decreased ENaC activity in oocytes expressing αβγ‐ENaC (n=10). Conversely, GSSG significantly increased whole cell current in oocytes expressing δ1βγ‐ENaC (n=12) and in δ2βγ‐ENaC expressing oocytes (n=13) immediately upon extracellular exposure. While both α and δ ENaC subunits are directly modified by GSSG at Cys thiol sites, the effect and mechanism of GSSG posttranslational modification of α vs δ expressing Na channels differed significantly in our studies. Heterologous expression of αβR566Xγ ENaC in oocytes indicate that GSSG proteolytically degrades classical αβγ ENaC. Whole cell recordings and biotinylation studies indicate that δ2 ENaC subunits increase net Na transport via insertion of new membrane protein, whereas GSSG plays no role in δ1 transport and insertion into the cell membrane.ConclusionWe found that αβγ ENaC activity is decreased by GSSG. Conversely δβγ ENaC activity is significantly increased by GSSG. This conclusion highlights the need to better understand the opposing translational effects of α vs δ ENaC subunits, as it may occur in human tissue. Additional studies are needed to better understand the molecular regulation and pathophysiological roles of α and δ ENaC subunits.Support or Funding InformationSupport for this project was made possible by the University of Utah Investigator Initiated Grant awarded to MNH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Orthogonality of Pyrrolysine tRNA in the Xenopus oocyte

Chemical aminoacylation of orthogonal tRNA allows for the genetic encoding of a wide range of synthetic amino acids without the need to evolve specific aminoacyl-tRNA synthetases. This method, when paired with protein expression in the Xenopus laevis oocyte expression system, can extract atomic scale functional data from a protein structure to advance the study of membrane proteins. The utility of the method depends on the orthogonality of the tRNA species used to deliver the amino acid. Here, we report that the pyrrolysyl tRNA (pylT) from Methanosarcina barkeri fusaro is orthogonal and highly competent for genetic code expansion experiments in the Xenopus oocyte. The data show that pylT is amendable to chemical acylation in vitro; it is then used to rescue a cytoplasmic site within a voltage-gated sodium channel. Further, the high fidelity of the pylT is demonstrated via encoding of lysine within the selectivity filter of the sodium channel, where sodium ion recognition by the distal amine of this side-chain is essential. Thus, pylT is an appropriate tRNA species for delivery of amino acids via nonsense suppression in the Xenopus oocyte. It may prove useful in experimental contexts wherein reacylation of suppressor tRNAs have been observed.