The Use of Xenopus laevis Oocytes for the Functional Characterization of Heterologously Expressed Membrane Proteins

2000 ◽  
Vol 10 (1-2) ◽  
pp. 1-12 ◽  
Author(s):  
Carsten A. Wagner ◽  
Björn Friedrich ◽  
Iwan Setiawan ◽  
Florian Lang ◽  
Stefan Bröer
Keyword(s):  
Membrane Proteins ◽  
Xenopus Laevis ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes

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

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.

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

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.

Molecular characterization of the murine Slc26a6 anion exchanger: functional comparison with Slc26a1

2002 ◽  
Vol 283 (4) ◽  
pp. F826-F838 ◽  
Author(s):  
Qizhi Xie ◽  
Rick Welch ◽  
Adriana Mercado ◽  
Michael F. Romero ◽  
David B. Mount
Keyword(s):  
Alternative Splicing ◽  
Xenopus Laevis ◽  
Proximal Tubule ◽  
Molecular Characterization ◽  
Anion Exchanger ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Membrane Hyperpolarization ◽  
Oxalate Transport

We report the molecular and functional characterization of murine Slc26a6, the putative apical chloride-formate exchanger of the proximal tubule. The Slc26a6 transcript is expressed in several tissues, including kidney. Alternative splicing of the second exon generates two distinct isoforms, denoted Slc26a6a and Slc26a6b, which differ in the inclusion of a 23-residue NH2-terminal extension. Functional comparison with murine Slc26a1, the basolateral oxalate exchanger of the proximal tubule, reveals a number of intriguing differences. Whereas Slc26a6 is capable of Cl−, SO[Formula: see text], formate, and oxalate uptake when expressed in Xenopus laevis oocytes, Slc26a1 transports only SO[Formula: see text] and oxalate. Measurement of intracellular pH during the removal of extracellular Cl− in the presence and absence of HCO[Formula: see text] indicates that Slc26a6 functions as both a Cl−/HCO[Formula: see text] and a Cl−/OH− exchanger; simultaneous membrane hyperpolarization during these experimental maneuvers reveals that HCO[Formula: see text] and OH− transport mediated by Slc26a6 is electrogenic. Cis-inhibition and efflux experiments indicate that Slc26a6 can mediate the exchange of both Cl− and SO[Formula: see text]with a number of substrates, including formate and oxalate. In contrast, SO[Formula: see text] and oxalate transport by Slc26a1 are mutually cis-inhibited but activated significantly by extracellular halides, lactate, and formate. The data indicate that Slc26a6 encodes an apical Cl−/formate/oxalate and Cl−/base exchanger and reveal significant mechanistic differences between apical and basolateral oxalate exchangers of the proximal tubule.

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

2021 ◽  
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.

scholarly journals Functional characterization of mouse urea transporters UT-A2 and UT-A3 expressed in purified Xenopus laevis oocyte plasma membranes

2008 ◽  
Vol 294 (4) ◽  
pp. F956-F964 ◽  
Author(s):  
Bryce MacIver ◽  
Craig P. Smith ◽  
Warren G. Hill ◽  
Mark L. Zeidel
Keyword(s):  
Xenopus Laevis ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Functional Characterization ◽  
Membrane Vesicles ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Plasma Membrane Vesicles ◽  
Biophysical Characterization

Urea is a small solute synthesized by many terrestrial organisms as part of the catabolism of protein. In mammals it is transported across cellular membranes by specific urea transporter (UT) proteins that are the products of two separate, but closely related genes, referred to as UT-A and UT-B. Three major UT-A isoforms are found in the kidney, namely UT-A1, UT-A2, and UT-A3. UT-A2 is found in the thin, descending limb of the loop of Henle, whereas UT-A1 and UT-A3 are concentrated in the inner medullary collecting duct. UT-A2 and UT-A3 effectively represent two halves of the whole UT-A gene and, when joined together by 73 hydrophilic amino acids, constitute UT-A1. A biophysical characterization of mouse UT-A2 and UT-A3 was undertaken by expression in Xenopus laevis oocytes and subsequent preparation of highly enriched plasma membrane vesicles for use in stopped-flow fluorometry. Both isoforms were found to be highly specific for urea, and did not permeate water, ammonia, or other molecules closely related to urea (formamide, acetamide, methylurea, and dimethylurea). Single transporter flux rates of 46,000 ± 10,000 and 59,000 ± 15,000 (means ± SE) urea molecules/s/channel for UT-A2 and UT-A3, respectively, were obtained. Overall, the UT-A2 and UT-A3 isoforms appear to have identical functional kinetics.

Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney

2000 ◽  
Vol 279 (3) ◽  
pp. F482-F490 ◽  
Author(s):  
Ana M. Pajor ◽  
Nina N. Sun
Keyword(s):  
Sequence Similarity ◽  
Genomic Structure ◽  
Functional Characterization ◽  
Krebs Cycle ◽  
Initial Step ◽  
Xenopus Laevis Oocytes ◽  
Chromosome 11 ◽  
Inward Currents ◽  
Urinary Citrate

The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations.1 Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na+-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is ∼24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant ( K m) for succinate was 0.35 mM, and the K m for citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at −50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity.

Functional characterization of human NBC4 as an electrogenic Na+-HCO 3 − cotransporter (NBCe2)

2002 ◽  
Vol 282 (6) ◽  
pp. C1278-C1289 ◽  
Author(s):  
Leila V. Virkki ◽  
Darren A. Wilson ◽  
Richard D. Vaughan-Jones ◽  
Walter F. Boron
Keyword(s):  
Human Heart ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Regulatory Function ◽  
Disulfonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Slow Recovery ◽  
Bicarbonate Transporter ◽  
Electrode Voltage

We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215–218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337 ) in Xenopus laevis oocytes and assayed membrane potential ( V m) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO[Formula: see text]elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pHi) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO[Formula: see text]- and Na+-dependent currents. When we voltage clamped V m far from NBC4c's estimated reversal potential ( E rev), the pHirecovery rate increased substantially. Both the currents and pHi recovery were blocked by 200 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). We estimated the transporter's HCO[Formula: see text]:Na+ stoichiometry by measuring E rev at different extracellular Na+ concentration ([Na+]o) values. A plot of E rev against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute E rev values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.

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