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.

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.

scholarly journals Characterization of the transport activity of SGLT2/MAP17, the renal low-affinity Na+-glucose cotransporter

2017 ◽  
Vol 313 (2) ◽  
pp. F467-F474 ◽  
Author(s):  
Michael J. Coady ◽  
Bernadette Wallendorff ◽  
Jean-Yves Lapointe
Keyword(s):  
Steady State ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Xenopus Laevis Oocytes ◽  
Electrogenic Transport ◽  
Affinity Constants ◽  
Glucose Reabsorption ◽  
Renal Glucose Reabsorption ◽  
External Glucose

The cotransporter SGLT2 is responsible for 90% of renal glucose reabsorption, and we recently showed that MAP17 appears to work as a required β-subunit. We report in the present study a detailed functional characterization of human SGLT2 in coexpression with human MAP17 in Xenopus laevis oocytes. Addition of external glucose generates a large inward current in the presence of Na, confirming an electrogenic transport mechanism. At a membrane potential of −50 mV, SGLT2 affinity constants for glucose and Na are 3.4 ± 0.4 and 18 ± 6 mM, respectively. The change in the reversal potential of the cotransport current as a function of external glucose concentration clearly confirms a 1:1 Na-to-glucose transport stoichiometry. SGLT2 is selective for glucose and α-methylglucose but also transports, to a lesser extent, galactose and 3- O-methylglucose. SGLT2 can be inhibited in a competitive manner by phlorizin ( Ki = 31 ± 4 nM) and by dapagliflozin ( Ki = 0.75 ± 0.3 nM). Similarly to SGLT1, SGLT2 can be activated by Na, Li, and protons. Pre-steady-state currents for SGLT2 do exist but are small in amplitude and relatively fast (a time constant of ~2 ms). The leak current defined as the phlorizin-sensitive current in the absence of substrate was extremely small in the case of SGLT2. In summary, in comparison with SGLT1, SGLT2 has a lower affinity for glucose, a transport stoichiometry of 1:1, very small pre-steady-state and leak currents, a 10-fold higher affinity for phlorizin, and an affinity for dapagliflozin in the subnanomolar range.

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.

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 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 PCFT/HCP1 as the molecular entity of the carrier-mediated intestinal folate transport system in the rat model

2008 ◽  
Vol 294 (3) ◽  
pp. G660-G668 ◽  
Author(s):  
Katsuhisa Inoue ◽  
Yasuhiro Nakai ◽  
Sayaka Ueda ◽  
Shunsuke Kamigaso ◽  
Kin-ya Ohta ◽  
...  
Keyword(s):  
Small Intestine ◽  
Transport System ◽  
Functional Characterization ◽  
Cellular Membrane ◽  
Molecular Entity ◽  
Xenopus Laevis Oocytes ◽  
Rat Small Intestine ◽  
Distribution Profile ◽  
Folate Transport

Proton-coupled folate transporter/heme carrier protein 1 (PCFT/HCP1) has recently been identified as a transporter that mediates the translocation of folates across the cellular membrane by a proton-coupled mechanism and suggested to be the possible molecular entity of the carrier-mediated intestinal folate transport system. To further clarify its role in intestinal folate transport, we examined the functional characteristics of rat PCFT/HCP1 (rPCFT/HCP1) expressed in Xenopus laevis oocytes and compared with those of the carrier-mediated folate transport system in the rat small intestine evaluated by using the everted tissue sacs. rPCFT/HCP1 was demonstrated to transport folate and methotrexate more efficiently at lower acidic pH and, as evaluated at pH 5.5, with smaller Michaelis constant ( Km) for the former (2.4 μM) than for the latter (5.7 μM), indicating its characteristic as a proton-coupled folate transporter that favors folate than methotrexate as substrate. rPCFT/HCP1-mediated folate transport was found to be inhibited by several but limited anionic compounds, such as sulfobromophthalein and sulfasalazine. All these characteristics of rPCFT/HCP1 were in agreement with those of carrier-mediated intestinal folate transport system, of which the Km values were 1.2 and 5.8 μM for folate and methotrexate, respectively, in the rat small intestine. Furthermore, the distribution profile of the folate transport system activity along the intestinal tract was in agreement with that of rPCFT/HCP1 mRNA. This study is the first to clone rPCFT/HCP1, and we successfully provided several lines of evidence that indicate its role as the molecular entity of the intestinal folate transport system.

Functional characterization of alpha- and beta-intercalated cell types in rabbit cortical collecting duct

1991 ◽  
Vol 261 (3) ◽  
pp. F377-F385 ◽  
Author(s):  
H. Furuya ◽  
M. D. Breyer ◽  
H. R. Jacobson
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Cortical Collecting Duct ◽  
Electrical Measurements ◽  
Collecting Ducts

Single-cell electrical measurements and spectrophotometric determinations of intracellular pH were used to determine unique features of alpha- and beta-intercalated cells (alpha-IC, beta-IC) in in vitro perfused rabbit cortical collecting ducts (CCD). pHi rose in alpha-IC and fell in beta-IC after bath Cl- removal. Luminal Cl- removal did not change pHi of alpha-IC, but pHi of beta-IC rose by 0.36 +/- 0.01 pH units. Cl- concentration-dependent recovery of beta-IC pHi revealed a Cl- Km of 18.7 mM for the luminal Cl(-) -HCO3- exchanger. Measurements of basolateral membrane voltage (Vbl) also showed two IC cell types. Removal of luminal Cl- did not change Vbl in alpha-IC, whereas Vbl hyperpolarized by a mean of 73.2 +/- 3.5 mV in beta-IC. Reducing bath Cl- depolarized both alpha- and beta-IC Vbl. In alpha-IC a large repolarization of 39.8 +/- 5.2 mV followed acute depolarization after bath Cl- removal. Reducing bath HCO3- (constant CO2) had little effect on beta-IC Vbl, whereas alpha-IC Vbl depolarized by 5.2 +/- 0.7 mV. Reducing luminal HCO3- in the absence of luminal Cl- produced a 17.6 +/- 1.8 mV depolarization in beta-IC. This change was independent of luminal Na+ and was not blocked by luminal 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In beta-IC, Vbl was not altered by either bath or lumen DIDS in the presence of luminal Cl-. However, when luminal Cl- was removed, luminal DIDS reversibly depolarized Vbl by 9.6 +/- 2.9 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Revisiting the functional properties of NPF6.3/NRT1.1/CHL1 in xenopus oocytes

2018 ◽  
Author(s):  
Mélanie Noguero ◽  
Sophie Léran ◽  
Eléonore Bouguyon ◽  
Chantal Brachet ◽  
Pascal Tillard ◽  
...  
Keyword(s):  
Transport Properties ◽  
Xenopus Oocytes ◽  
Nitrate Concentration ◽  
Functional Characterization ◽  
Nitrate Transport ◽  
Experimental Conditions ◽  
Point Mutant ◽  
Electrode Voltage ◽  
Nitrate Transporters

ABSTRACTWithin the Arabidopsis NPF proteins, most of the characterized nitrate transporters are low-affinity transporters, whereas the functional characterization of NPF6.3/NRT1.1 has revealed interesting transport properties: the transport of nitrate and auxin, the eletrogenicity of the nitrate transport and a dual-affinity transport behavior for nitrate depending on external nitrate concentration. However, some of these properties remained controversial and were challenged here. We functionally express WT NPF6.3/NRT1.1 and some of its mutant in Xenopus oocytes and used a combination of uptake experiments using 15N-labelled nitrate and two-electrode voltage-clamp. In our experimental conditions in xenopus oocytes, in the presence or in the absence of external chloride, NPF6.3/NRT1.1 behaves as a non-electrogenic and pure low-affinity transporter. Moreover, further functional characterization of a NPF6.3/NRT1.1 point mutant, P492L, allowed us to hypothesize that NPF6.3/NRT1.1 is regulated by internal nitrate concentration and that the internal perception site involves the P492 residue.

NH2-terminal heterogeneity in the KCC3 K+-Cl− cotransporter

2005 ◽  
Vol 289 (6) ◽  
pp. F1246-F1261 ◽  
Author(s):  
Adriana Mercado ◽  
Norma Vázquez ◽  
Luyan Song ◽  
Rosa Cortés ◽  
Alissa H. Enck ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Functional Characterization ◽  
Cell Volume Regulation ◽  
Cell Swelling ◽  
Mouse Tissue ◽  
Xenopus Laevis Oocytes ◽  
Acceptor Site ◽  
Exon 2 ◽  
Mouse Tissues

The SLC12A6 gene encoding the K+-Cl− cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH2-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5′ of two distinct first coding exons. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both the mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of NH2-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in X. laevis oocytes reveals apparent Kms for Rb+ and Cl− of 10.7 ± 2.5 and 7.3 ± 1.2 mM, respectively, with an anion selectivity of Br− > Cl− > PO4 = I− = SCN− = gluconate. All five NH2-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S proteins. In summary, there is significant NH2-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the kidney. Basolateral swelling-activated K+-Cl− cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.

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