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

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.

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Genomic structure, expression pattern, and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon)

PLoS ONE ◽

10.1371/journal.pone.0177420 ◽

2017 ◽

Vol 12 (5) ◽

pp. e0177420 ◽

Cited By ~ 2

Author(s):

Bobo Xie ◽

Pengfei Wang ◽

Chao Zhao ◽

Lihua Qiu

Keyword(s):

Transcription Factor ◽

Expression Pattern ◽

Genomic Structure ◽

Functional Characterization ◽

Penaeus Monodon ◽

Black Tiger Shrimp ◽

Tiger Shrimp ◽

Transcription Factor E2f

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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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An aphid effector promotes barley susceptibility through suppression of defence gene expression

Journal of Experimental Botany ◽

10.1093/jxb/eraa043 ◽

2020 ◽

Vol 71 (9) ◽

pp. 2796-2807 ◽

Cited By ~ 1

Author(s):

Carmen Escudero-Martinez ◽

Patricia A Rodriguez ◽

Shan Liu ◽

Pablo A Santos ◽

Jennifer Stephens ◽

...

Keyword(s):

Gene Expression ◽

Sequence Similarity ◽

Rhopalosiphum Padi ◽

Functional Characterization ◽

Aphid Species ◽

Transgenic Barley ◽

Defence Gene Expression ◽

Hormone Signalling ◽

Plant Susceptibility

Abstract Aphids secrete diverse repertoires of effectors into their hosts to promote the infestation process. While ‘omics’ approaches facilitated the identification and comparison of effector repertoires from a number of aphid species, the functional characterization of these proteins has been limited to dicot (model) plants. The bird cherry-oat aphid Rhopalosiphum padi is a pest of cereal crops, including barley. Here, we extend efforts to characterize aphid effectors with regard to their role in promoting susceptibility to the R. padi–barley interaction. We selected three R. padi effectors based on sequence similarity to previously characterized Myzus persicae effectors and assessed their subcellular localization, expression, and role in promoting plant susceptibility. Expression of R. padi effectors RpC002 and Rp1 in transgenic barley lines enhanced plant susceptibility to R. padi but not M. persicae, for which barley is a poor host. Characterization of Rp1 transgenic barley lines revealed reduced gene expression of plant hormone signalling genes relevant to plant–aphid interactions, indicating that this effector enhances susceptibility by suppressing plant defences in barley. Our data suggest that some aphid effectors specifically function when expressed in host species, and feature activities that benefit their corresponding aphid species.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00589.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. C1278-C1289 ◽

Cited By ~ 90

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

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00309.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. G660-G668 ◽

Cited By ~ 67

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.

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The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants

Pharmacogenetics ◽

10.1097/00008571-200110000-00003 ◽

2001 ◽

Vol 11 (7) ◽

pp. 555-572 ◽

Cited By ~ 219

Author(s):

Jiong Zhang ◽

Peter Kuehl ◽

Eric D. Green ◽

Jeffery W. Touchman ◽

Paul B. Watkins ◽

...

Keyword(s):

Genomic Structure ◽

Functional Characterization ◽

Pregnane X Receptor ◽

Allelic Variants

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Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation

Journal of Bacteriology ◽

10.1128/jb.01318-07 ◽

2007 ◽

Vol 189 (22) ◽

pp. 8088-8098 ◽

Cited By ~ 102

Author(s):

Amirreza Faridmoayer ◽

Messele A. Fentabil ◽

Dominic C. Mills ◽

John S. Klassen ◽

Mario F. Feldman

Keyword(s):

Sequence Similarity ◽

Functional Characterization ◽

Protein Glycosylation ◽

Structural Components ◽

En Bloc ◽

E Coli ◽

New Family ◽

Type Iv ◽

Domains Of Life

ABSTRACT Protein glycosylation is an important posttranslational modification that occurs in all domains of life. Pilins, the structural components of type IV pili, are O glycosylated in Neisseria meningitidis, Neisseria gonorrhoeae, and some strains of Pseudomonas aeruginosa. In this work, we characterized the P. aeruginosa 1244 and N. meningitidis MC58 O glycosylation systems in Escherichia coli. In both cases, sugars are transferred en bloc by an oligosaccharyltransferase (OTase) named PglL in N. meningitidis and PilO in P. aeruginosa. We show that, like PilO, PglL has relaxed glycan specificity. Both OTases are sufficient for glycosylation, but they require translocation of the undecaprenol-pyrophosphate-linked oligosaccharide substrates into the periplasm for activity. Whereas PilO activity is restricted to short oligosaccharides, PglL is able to transfer diverse oligo- and polysaccharides. This functional characterization supports the concept that despite their low sequence similarity, PilO and PglL belong to a new family of “O-OTases” that transfer oligosaccharides from lipid carriers to hydroxylated amino acids in proteins. To date, such activity has not been identified for eukaryotes. To our knowledge, this is the first report describing recombinant O glycoproteins synthesized in E. coli.

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