Expression and Ion Transport Activity of Rice OsHKT1;1 Variants

OsHKT1;1 in rice, belongs to the high-affinity K+ Transporter family, has been found to be involved in salt tolerance. OsHKT1;1 in japonica rice (Nipponbare) produces mRNA variants, but their functions remain elusive. In salt tolerant rice, Pokkali, eight OsHKT1;1 variants (V1-V8) were identified in addition to the full-length OsHKT1;1 (FL) cDNA. Absolute quantification by qPCR revealed that accumulation of OsHKT1;1-FL mRNA is minor in contrast to that of OsHKT1;1-V1, -V2, -V4, and -V7 mRNAs, all of which are predominant in shoots, while only V1 and V7 mRNAs are predominant in roots. Two electrode voltage clamp (TEVC) experiments using Xenopus laevis oocytes revealed that oocytes-expressing OsHKT1;1-FL from Pokkali exhibited inward-rectified currents in the presence of 96 mM Na+ as reported previously. Further TEVC analyses indicated that six of eight OsHKT1;1 variants elicited currents in a Na+ or a K+ bath solution. OsHKT1;1-V6 exhibited a similar inward rectification to the FL protein. Contrastingly, however, the rests mediated bidirectional currents in both Na+ and K+ bath solutions. These data suggest possibilities that novel mechanisms regulating the transport activity of OsHKT1;1 might exist, and that OsHKT1;1 variants might also carry out distinct physiological roles either independently or in combination with OsHKT1;1-FL.

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Identification and Characterization of Rice OsHKT1;3 Variants

Plants ◽

10.3390/plants10102006 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2006

Author(s):

Shahin Imran ◽

Yoshiyuki Tsuchiya ◽

Sen Thi Huong Tran ◽

Maki Katsuhara

Keyword(s):

Expression System ◽

Ion Homeostasis ◽

Transcript Level ◽

Absolute Quantification ◽

Xenopus Laevis Oocytes ◽

Japonica Rice ◽

Na Currents ◽

Mrna Variants ◽

Identification And Characterization

In rice, the high-affinity K+ transporter, OsHKT1;3, functions as a Na+-selective transporter. mRNA variants of OsHKT1;3 have been reported previously, but their functions remain unknown. In this study, five OsHKT1;3 variants (V1-V5) were identified from japonica rice (Nipponbare) in addition to OsHKT1;3_FL. Absolute quantification qPCR analyses revealed that the transcript level of OsHKT1;3_FL was significantly higher than other variants in both the roots and shoots. Expression levels of OsHKT1;3_FL, and some variants, increased after 24 h of salt stress. Two electrode voltage clamp experiments in a heterologous expression system using Xenopus laevis oocytes revealed that oocytes expressing OsHKT1;3_FL and all of its variants exhibited smaller Na+ currents. The presented data, together with previous data, provide insights to understanding how OsHKT family members are involved in the mechanisms of ion homeostasis and salt tolerance in rice.

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Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211004123 ◽

2021 ◽

pp. 247255522110041

Author(s):

Raffaella Cinquetti ◽

Francesca Guia Imperiali ◽

Salvatore Bozzaro ◽

Daniele Zanella ◽

Francesca Vacca ◽

...

Keyword(s):

Xenopus Laevis ◽

Voltage Clamp ◽

Expression System ◽

Peptide Transporter ◽

Xenopus Laevis Oocytes ◽

Substrate Uptake ◽

Transport Activity ◽

Experimental Conditions ◽

Metal Transporters ◽

Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

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ScOPT1 and AtOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities

Biochemical Journal ◽

10.1042/bj20050920 ◽

2005 ◽

Vol 393 (1) ◽

pp. 267-275 ◽

Cited By ~ 44

Author(s):

Hiroki Osawa ◽

Gary Stacey ◽

Walter Gassmann

Keyword(s):

Xenopus Laevis Oocytes ◽

Transport Mechanisms ◽

Biological Functions ◽

Yeast Growth ◽

Substrate Specificities ◽

High Affinity ◽

Electrode Voltage ◽

Inward Currents ◽

Derivatives Of ◽

Substrate Concentrations

A group of OPTs (oligopeptide transporters) exclusively identified in plants and fungi are proposed to transport oligopeptides and derivatives of three to six amino acids in length, but their transport mechanisms and biological functions are poorly understood. We expressed the Saccharomyces cerevisiae (yeast) OPT ScOPT1 and five Arabidopsis thaliana AtOPTs in Xenopus laevis oocytes for two-electrode voltage-clamp studies. ScOPT1 produced inward currents in response to GSH or GSSG, the phytochelatin (PC) PC2 and oligopeptides including the tetrapeptide GGFL, but not KLGL. Inward currents were dependent on the external proton and substrate concentrations, with high affinity for both. This and the inward currents evoked by substrates with net negative charges showed that ScOPT1 is a proton-coupled transporter. ScOPT1 displayed highest apparent affinity for PC2, with small differences in the maximal current among substrates. Glutathione transport by any of the tested AtOPTs, including AtOPT6, was not detected in yeast growth complementation assays. With AtOPT4, initially only small KLGL-dependent currents were recorded in batches of oocytes showing high ScOPT1 expression. AtOPT4 expression was optimized by swapping the 5′-untranslated region with that of ScOPT1. AtOPT4 displayed a higher affinity for KLGL than ScOPT1 did for any peptide. AtOPT4-mediated KLGL transport was detectable at pH 5.0, but not at pH 6.0 or 7.0. Taken together, our results demonstrate that ScOPT1 and AtOPT4 are proton-coupled OPTs with broad but distinct substrate specificities and affinities.

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Two oligopeptide transporters from Caenorhabditis elegans:molecular cloning and functional expression

Biochemical Journal ◽

10.1042/bj3320565 ◽

1998 ◽

Vol 332 (2) ◽

pp. 565-572 ◽

Cited By ~ 31

Author(s):

You-Jun FEI ◽

Takuya FUJITA ◽

David F. LAPP ◽

Vadivel GANAPATHY ◽

Frederick H. LEIBACH

Keyword(s):

Amino Acid ◽

Functional Expression ◽

Amino Acid Sequences ◽

Xenopus Laevis Oocytes ◽

Cdna Clones ◽

Transport Activity ◽

Animal Kingdom ◽

Peptide Substrates ◽

Electrogenic Transport ◽

High Affinity

Two novel oligopeptide transporter cDNA clones, CPTA and CPTB, were identified by screening a Caenorhabditis elegans cDNA library using homology hybridization. The transporter proteins deduced from the cDNAs possess multiple transmembrane domains and reveal a moderate similarity to their mammalian counterparts in amino acid sequences. CPTA and CPTB, when expressed in Xenopus laevis oocytes and studied by both radiotracer flux and microelectrode voltage-clamp protocol, displayed a saturable electrogenic transport activity driven by a proton gradient with an overlapping broad spectrum of substrate specificity. Both transporters recognize di-, tri- and tetra-peptides including phenylalanylmethionylarginylphenylalaninamide (FMRFamide) and N-acetylaspartylglutamate, members of a large neuropeptide family commonly found throughout the animal kingdom. Kinetic analysis, however, revealed that CPTA and CPTB differed in their affinity for the peptide substrates, the former being a high-affinity type and the latter a low-affinity type. CPTA and CPTB are encoded by two distinct genes localized on separate chromosomes and are expressed during the whole life span of the organism.

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The SLC1 high-affinity glutamate and neutral amino acid transporter family

Molecular Aspects of Medicine ◽

10.1016/j.mam.2013.01.001 ◽

2013 ◽

Vol 34 (2-3) ◽

pp. 108-120 ◽

Cited By ~ 147

Author(s):

Yoshikatsu Kanai ◽

Benjamin Clémençon ◽

Alexandre Simonin ◽

Michele Leuenberger ◽

Martin Lochner ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Transporter ◽

Neutral Amino Acid ◽

High Affinity ◽

Neutral Amino Acid Transporter ◽

Transporter Family ◽

Acid Transporter

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Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris

Pharmaceuticals ◽

10.3390/ph14070698 ◽

2021 ◽

Vol 14 (7) ◽

pp. 698

Author(s):

Tina V. A. Hansen ◽

Richard K. Grencis ◽

Mohamed Issouf ◽

Cédric Neveu ◽

Claude L. Charvet

Keyword(s):

Single Dose ◽

Mouse Model ◽

Xenopus Laevis ◽

Acetylcholine Receptor ◽

Drug Target ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Trichuris Muris ◽

Electrode Voltage

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

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Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00075.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. G271-G278 ◽

Cited By ~ 77

Author(s):

Chitrawina Mahagita ◽

Steven M. Grassl ◽

Pawinee Piyachaturawat ◽

Nazzareno Ballatori

Keyword(s):

Bile Acid ◽

Transport Mechanism ◽

Organic Anion ◽

Large Family ◽

Organic Anion Transporter ◽

Xenopus Laevis Oocytes ◽

Facilitated Diffusion ◽

Transport Activity ◽

Estrone Sulfate ◽

Anion Transporter

Organic anion transporting polypeptides (OATP/ SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 ( Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 ( SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C ( SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.

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Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity

Biochemical Journal ◽

10.1042/bj20020084 ◽

2002 ◽

Vol 364 (3) ◽

pp. 767-775 ◽

Cited By ~ 40

Author(s):

Sabine WOLF ◽

Annette JANZEN ◽

Nicole VÉKONY ◽

Ursula MARTINÉ ◽

Dennis STRAND ◽

...

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Protein Expression ◽

Amino Acid Transport ◽

Fluorescent Protein ◽

Xenopus Laevis Oocytes ◽

Amino Acid Transporters ◽

Acid Transport ◽

Transport Activity ◽

Solute Carrier

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230–236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional.

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The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel

Molecules ◽

10.3390/molecules27010312 ◽

2022 ◽

Vol 27 (1) ◽

pp. 312

Author(s):

Tina V. A. Hansen ◽

Heinz Sager ◽

Céline E. Toutain ◽

Elise Courtot ◽

Cédric Neveu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Parasitic Nematode ◽

Nematode Species ◽

Xenopus Laevis Oocytes ◽

Allosteric Modulators ◽

C Elegans ◽

Electrode Voltage ◽

Insight Into

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

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Regulation of intestinal phosphate cotransporter NaPi IIb by ubiquitin ligase Nedd4–2 and by serum- and glucocorticoid-dependent kinase 1

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00121.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. G143-G150 ◽

Cited By ~ 41

Author(s):

M. Palmada ◽

M. Dieter ◽

A. Speil ◽

C. Böhmer ◽

A. F. Mack ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Phosphate Transport ◽

Transport Systems ◽

Xenopus Laevis Oocytes ◽

Intestinal Epithelial ◽

Induced Current ◽

Intestinal Function ◽

Transport Activity ◽

Wild Type ◽

Subsequent Degradation

Serum and glucocorticoid-inducible kinase 1 (SGK1) is highly expressed in enterocytes. The significance of the kinase in regulation of intestinal function has, however, remained elusive. In Xenopus laevis oocytes, SGK1 stimulates the epithelial Na+ channel by phosphorylating the ubiquitin ligase Nedd4–2, which regulates channels by ubiquitination leading to subsequent degradation of the channel protein. Thus the present study has been performed to explore whether SGK1 regulates transport systems expressed in intestinal epithelial cells, specifically type IIb sodium-phosphate (Na+-Pi) cotransporter (NaPi IIb). Immunohistochemistry in human small intestine revealed SGK1 colocalization with Nedd4–2 in villus enterocytes. For functional analysis cRNA encoding NaPi IIb, the SGK isoforms and/or the Nedd4–2 were injected into X. laevis oocytes, and transport activity was quantified as the substrate-induced current ( IP). Exposure to 3 mM phosphate induces an IP in NaPi IIb-expressing oocytes. Coinjection of Nedd4–2, but not the catalytically inactive mutant C938SNedd4–2, significantly downregulates IP, whereas the coinjection of S422DSGK1 markedly stimulates IP and even fully reverses the effect of Nedd4–2 on IP. The effect of S422DSGK1 on NaPi IIb is mimicked by wild-type SGK3 but not by wild-type SGK2, constitutively active T308D,S473DPKB, or inactive K127NSGK1. Moreover, S422DSGK1 and SGK3 phosphorylate Nedd4–2. In conclusion, SGK1 stimulates the NaPi IIb, at least in part, by phosphorylating and thereby inhibiting Nedd4–2 binding to its target. Thus the present study reveals a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for regulation of phosphate balance.

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