Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)-expressing Xenopus laevis oocytes by using pH-sensitive microelectrodes and [14C]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, d,l-3-hydroxybutyrate, acetoacetate, α-oxoisohexanoate and α-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor α-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.

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Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

Biochemical Journal ◽

10.1042/bj3410529 ◽

1999 ◽

Vol 341 (3) ◽

pp. 529-535 ◽

Cited By ~ 131

Author(s):

Stefan BRÖER ◽

Angelika BRÖER ◽

Hans-Peter SCHNEIDER ◽

Carola STEGEN ◽

Andrew P. HALESTRAP ◽

...

Keyword(s):

Ketone Bodies ◽

Anion Channel ◽

Monocarboxylate Transporter ◽

Xenopus Laevis Oocytes ◽

Lactate Transport ◽

High Affinity ◽

Monocarboxylate Transport ◽

Km Value ◽

Decreasing Ph

Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevisoocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H+ gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, β-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a Km value of 0.74±0.07 mM was determined at pH 7.0. For the other substrates, Ki values between 100 μM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by α-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.

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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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Functional characterization of leucine transport induced in Xenopus laevis oocytes injected with mRNA isolated from midguts of lepidopteran larvae (Philosamia cynthia).

Journal of Experimental Biology ◽

10.1242/jeb.198.4.961 ◽

1995 ◽

Vol 198 (4) ◽

pp. 961-966

Author(s):

V F Sacchi ◽

C Perego ◽

S Magagnin

Keyword(s):

Xenopus Laevis ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Fourfold Increase ◽

Leucine Uptake ◽

Lepidopteran Larvae ◽

Nacl Concentration ◽

Leucine Transport ◽

The Difference

The injection of poly(A)+ mRNA prepared from Philosamia cynthia midgut caused time- and dose-dependent increases of leucine transport in Xenopus laevis oocytes, with an increase in leucine uptake 1.5-3 times that of oocytes injected with water. When the NaCl concentration was reduced from 100 to 5 mmol l-1, the difference between mRNA- and water-injected oocytes was greater and a fourfold increase of L-leucine uptake was measured. D-Leucine (10 mmol l-1) completely inhibited the induced uptake of 0.1 mmol l-1 L-leucine. The newly expressed component of L-leucine uptake increased at alkaline pH and was abolished by incubation for 15 min with 15 mmol l-1 phenylglyoxal. The mean Km values, calculated using Na+ activation curves of leucine uptake, were 23.3 +/- 6.1 mmol l-1 in water-injected oocytes and 0.4 +/- 0.2 mmol l-1 for the newly expressed component of leucine uptake in mRNA-injected oocytes. On the basis of these results, we conclude that the increase of L-leucine uptake in mRNA-injected oocytes was due to the expression of a new transport system, which differs from the endogenous ones and shares many features with that found previously in Philosamia cynthia midgut.

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Assessment of natural variation in the first pore domain of the tomato HKT1;2 transporter and characterization of mutated versions of SlHKT1;2 expressed in Xenopus laevis oocytes and via complementation of the salt sensitive athkt1;1 mutant

Frontiers in Plant Science ◽

10.3389/fpls.2014.00600 ◽

2014 ◽

Vol 5 ◽

Cited By ~ 7

Author(s):

Pedro M. F. Almeida ◽

Gert-Jan de Boer ◽

Albertus H. de Boer

Keyword(s):

Xenopus Laevis ◽

Natural Variation ◽

Xenopus Laevis Oocytes ◽

Pore Domain

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Functional characterization of high-affinity Na+/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

AJP Cell Physiology ◽

10.1152/ajpcell.00295.2004 ◽

2005 ◽

Vol 289 (5) ◽

pp. C1159-C1168 ◽

Cited By ~ 9

Author(s):

Naomi Oshiro ◽

Ana M. Pajor

Keyword(s):

Xenopus Laevis ◽

Cdna Sequence ◽

Citric Acid Cycle ◽

Functional Characterization ◽

Activation Kinetics ◽

Electrophysiological Properties ◽

High Affinity ◽

The Family ◽

Kidney Liver

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na+/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is ∼70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a Km for succinate of 4 μM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site.

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Rapid downregulation of the rat glutamine transporter SNAT3 by a caveolin-dependent trafficking mechanism in Xenopus laevis oocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00209.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. C1047-C1057 ◽

Cited By ~ 15

Author(s):

Sarojini Balkrishna ◽

Angelika Bröer ◽

Alice Kingsland ◽

Stefan Bröer

Keyword(s):

Xenopus Laevis ◽

Dominant Negative ◽

Monocarboxylate Transporter ◽

Xenopus Laevis Oocytes ◽

Independent Manner ◽

Cultured Astrocytes ◽

Glutamine Transporter ◽

Liver And Kidney ◽

The Brain ◽

Glutamine Uptake

The glutamine transporter SNAT3 is involved in the uptake and release of glutamine in the brain, liver, and kidney. Substrate transport is accompanied by Na+ cotransport and H+ antiport. In this study, treatment of Xenopus laevis oocytes expressing rat SNAT3 with the phorbol ester PMA resulted in a rapid downregulation of glutamine uptake in less than 20 min. PMA treatment of oocytes coexpressing SNAT3 and the monocarboxylate transporter MCT1 reduced SNAT3 activity only, demonstrating the specificity of the regulatory mechanism. Single or combined mutations of seven putative phosphorylation sites in the SNAT3 sequence did not affect the regulation of SNAT3 by PMA. Expression of an EGFP-SNAT3 fusion protein in oocytes established that the downregulation was caused by the retrieval of the transporter from the plasma membrane. Coexpression of SNAT3 with dominant-negative mutants of dynamin or caveolin revealed that SNAT3 trafficking occurs in a dynamin-independent manner and is influenced by caveolin. Although system N activity was not affected by PMA in cultured astrocytes, a downregulation was observed in HepG2 cells.

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