Functional characterization of testis-specific rodent multidrug and toxic compound extrusion 2, a class III MATE-type polyspecific H+/organic cation exporter

Mammalian multidrug and toxic compound extrusion (MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H+-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney (Otsuka et al. Proc Natl Acad Sci USA 102: 17923–17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587–593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H+-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K+. Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H+/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations.

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Wide variety of locations for rodent MATE1, a transporter protein that mediates the final excretion step for toxic organic cations

AJP Cell Physiology ◽

10.1152/ajpcell.00090.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. C678-C686 ◽

Cited By ~ 71

Author(s):

Miki Hiasa ◽

Takuya Matsumoto ◽

Toshinori Komatsu ◽

Yoshinori Moriyama

Keyword(s):

Transport Properties ◽

Leydig Cells ◽

Wide Distribution ◽

The Body ◽

Organic Cations ◽

Bladder Epithelium ◽

Ito Cells ◽

Hek 293 ◽

Transporter Protein ◽

293 Cells

MATE1 was the first mammalian example of the multidrug and toxin extrusion (MATE) protein family to be identified. Human MATE1 (hMATE1) is predominantly expressed and localized to the luminal membranes of the urinary tubules and bile canaliculi and mediates H+-coupled electroneutral excretion of toxic organic cations (OCs) into urine and bile (Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, and Moriyama Y. Proc Natl Acad Sci USA 102: 17923–17928, 2005). mMATE1, a mouse MATE ortholog, is also predominantly expressed in kidney and liver, although its transport properties are not yet characterized. In the present study, we investigated the transport properties and localization of mMATE1. Upon expression of this protein in HEK-293 cells, mMATE1 mediated electroneutral H+/tetraethylammonium exchange and showed a substrate specificity similar to that of hMATE1. Immunological techniques with specific antibodies against mMATE1 combined with RT-PCR revealed that mMATE1 is also expressed in various cells, including brain glia-like cells and capillaries, pancreatic duct cells, urinary bladder epithelium, adrenal gland cortex, α cells of the islets of Langerhans, Leydig cells, and vitamin A-storing Ito cells. These results indicate that mMATE1 is a polyspecific H+/OC exchanger. The unexpectedly wide distribution of mMATE1 suggests involvement of this transporter protein in diverse biological functions other than excretion of OCs from the body.

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The TRPV3 channel of the bovine rumen: localization and functional characterization of a protein relevant for ruminal ammonia transport

Pflügers Archiv - European Journal of Physiology ◽

10.1007/s00424-020-02393-2 ◽

2020 ◽

Vol 472 (6) ◽

pp. 693-710 ◽

Cited By ~ 2

Author(s):

Franziska Liebe ◽

Hendrik Liebe ◽

Sabine Kaessmeyer ◽

Gerhard Sponder ◽

Friederike Stumpff

Keyword(s):

Single Channel ◽

Functional Characterization ◽

Nitrogen Efficiency ◽

Channel Measurements ◽

Hek 293 ◽

Bovine Rumen ◽

Ruminal Ph ◽

293 Cells ◽

Ammonia Transport ◽

Ruminal Epithelium

Abstract Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed.

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Functional Characterization of the Regulatory Region of Human CD2-Associated Protein Promoter in HEK 293 Cells

American Journal of Nephrology ◽

10.1159/000155658 ◽

2009 ◽

Vol 29 (3) ◽

pp. 203-212 ◽

Cited By ~ 4

Author(s):

Xin-Ming Su ◽

Wei Ren ◽

Chao Lu ◽

Ji-Qing Chen ◽

Sheng-Hua Wu ◽

...

Keyword(s):

Functional Characterization ◽

Regulatory Region ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Cloning, expression, and functional characterization of the von Willebrand factor–cleaving protease (ADAMTS13)

Blood ◽

10.1182/blood-2002-05-1397 ◽

2002 ◽

Vol 100 (10) ◽

pp. 3626-3632 ◽

Cited By ~ 161

Author(s):

Barbara Plaimauer ◽

Klaus Zimmermann ◽

Dirk Völkel ◽

Gerhard Antoine ◽

Randolf Kerschbaumer ◽

...

Keyword(s):

Von Willebrand Factor ◽

Functional Activity ◽

Functional Characterization ◽

Eukaryotic Expression ◽

Gene Encoding ◽

Hek 293 ◽

293 Cells ◽

Von Willebrand ◽

Willebrand Factor

Deficient von Willebrand factor (VWF) degradation has been associated with thrombotic thrombocytopenic purpura (TTP). In hereditary TTP, the specific VWF-cleaving protease (VWF-cp) is absent or functionally defective, whereas in the nonfamilial, acquired form of TTP, an autoantibody inhibiting VWF-cp activity is found transiently in most patients. The gene encoding for VWF-cp has recently been identified as a member of the metalloprotease family and designatedADAMTS13, but the functional activity of the ADAMTS13 gene product has not been verified. To establish the functional activity of recombinant VWF-cp, we cloned the complete cDNA sequence in a eukaryotic expression vector and transiently expressed the encoded recombinant ADAMTS13 in HEK 293 cells. The expressed protein degraded VWF multimers and proteolytically cleaved VWF to the same fragments as those generated by plasma VWF-cp. Furthermore, recombinant ADAMTS13-mediated degradation of VWF multimers was entirely inhibited in the presence of plasma from a patient with acquired TTP. These data show that ADAMTS13 is responsible for the physiologic proteolytic degradation of VWF multimers.

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Clinical and Functional Characterization of the Recurrent TUBA1A p.(Arg2His) Mutation

Brain Sciences ◽

10.3390/brainsci8080145 ◽

2018 ◽

Vol 8 (8) ◽

pp. 145 ◽

Cited By ~ 4

Author(s):

Jennifer Gardner ◽

Thomas Cushion ◽

Georgios Niotakis ◽

Heather Olson ◽

P. Grant ◽

...

Keyword(s):

De Novo ◽

Functional Characterization ◽

Clinical Manifestations ◽

Fetal Brain ◽

Detailed Comparison ◽

Hek 293 ◽

293 Cells ◽

Brain Malformations ◽

Computer Based

The TUBA1A gene encodes tubulin alpha-1A, a protein that is highly expressed in the fetal brain. Alpha- and beta-tubulin subunits form dimers, which then co-assemble into microtubule polymers: dynamic, scaffold-like structures that perform key functions during neurogenesis, neuronal migration, and cortical organisation. Mutations in TUBA1A have been reported to cause a range of brain malformations. We describe four unrelated patients with the same de novo missense mutation in TUBA1A, c.5G>A, p.(Arg2His), as found by next generation sequencing. Detailed comparison revealed similar brain phenotypes with mild variability. Shared features included developmental delay, microcephaly, hypoplasia of the cerebellar vermis, dysplasia or thinning of the corpus callosum, small pons, and dysmorphic basal ganglia. Two of the patients had bilateral perisylvian polymicrogyria. We examined the effects of the p.(Arg2His) mutation by computer-based protein structure modelling and heterologous expression in HEK-293 cells. The results suggest the mutation subtly impairs microtubule function, potentially by affecting inter-dimer interaction. Based on its sequence context, c.5G>A is likely to be a common recurrent mutation. We propose that the subtle functional effects of p.(Arg2His) may allow for other factors (such as genetic background or environmental conditions) to influence phenotypic outcome, thus explaining the mild variability in clinical manifestations.

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SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization

AJP Cell Physiology ◽

10.1152/ajpcell.00089.2011 ◽

2011 ◽

Vol 301 (2) ◽

pp. C289-C303 ◽

Cited By ~ 32

Author(s):

Andrew K. Stewart ◽

Boris E. Shmukler ◽

David H. Vandorpe ◽

Fabian Reimold ◽

John F. Heneghan ◽

...

Keyword(s):

Guinea Pig ◽

Pancreatic Duct ◽

Xenopus Oocytes ◽

Functional Characterization ◽

Pancreatic Ducts ◽

Anion Exchangers ◽

Hek 293 ◽

Hek 293 Cells ◽

Anion Transporters ◽

293 Cells

The secretin-stimulated human pancreatic duct secretes HCO3−-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO3− secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl−/HCO3− exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO3− or more, mouse and rat ducts secrete ∼40–70 mM HCO3−. Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO3− secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl−/Cl− exchange and electroneutral Cl−/HCO3− exchange. gpSlc26a6 in Xenopus oocytes mediated Cl−/Cl− exchange and bidirectional exchange of Cl− for oxalate and sulfate, but Cl−/HCO3− exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl−, oxalate, and sulfate transport but no detectable Cl−/HCO3− exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of 36Cl− influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO3− secretion in species that share a high HCO3− secretory output.

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Involvement of the Multidrug and Toxic Compound Extrusion Transporter in Testosterone Release from Cultured Pig Leydig Cells

Pharmacology ◽

10.1159/000460822 ◽

2017 ◽

Vol 100 (1-2) ◽

pp. 31-39 ◽

Cited By ~ 1

Author(s):

Mitsuhiro Goda ◽

Kana Oda ◽

Atsuko Oda ◽

Naoki Kobayashi ◽

Masato Otsuka

Keyword(s):

Chorionic Gonadotropin ◽

Leydig Cells ◽

Physiological Function ◽

Immunohistochemical Analysis ◽

Organic Cations ◽

Toxic Compound ◽

Testosterone Levels ◽

Testosterone Secretion ◽

Cimetidine Treatment ◽

Mate Transporter

Testosterone is considered to be released from Leydig cells via passive diffusion because of its hydrophobicity; however, the exact mechanism underlying testosterone secretion and the transporter involved are both unknown. Multidrug and toxic compound extrusion (MATE) transporters are predominantly found in the kidneys and liver and are thought to function in the elimination of metabolic organic cations during the final step of excretion in the kidney. In contrast, mMATE2 has been shown to be predominantly expressed in testicular Leydig cells. Although the physiological function of mMATE2 in Leydig cells is unknown, we hypothesized that mMATE2 acts as a testosterone exporter and is responsible for the secretion of testosterone from Leydig cells. Therefore, in the present study, we investigated the involvement of the MATE transporter in testosterone secretion from pig Leydig cells. Immunohistochemical analysis with anti-pig MATE2 antiserum indicated that the MATE transporter is present in pig Leydig cells. Additionally, treatment with the MATE inhibitors cimetidine and pyrimethamine reduced the testosterone secretion from pig Leydig cells but increased the intracellular testosterone levels. Estradiol release and intracellular estradiol level induced by human chorionic gonadotropin (hCG) further increased with cimetidine treatment. These results indicated that testosterone produced by hCG treatment is secreted from Leydig cells via the MATE transporter; however, in the presence of cimetidine or pyrimethamine, this MATE transporter-mediated secretion was inhibited, resulting in increased intracellular testosterone levels and estradiol production in Leydig cells. Thus, the MATE transporter may be responsible for testosterone secretion from Leydig cells.

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Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.6.c1210 ◽

1999 ◽

Vol 277 (6) ◽

pp. C1210-C1219 ◽

Cited By ~ 149

Author(s):

Joanne E. Race ◽

Fadi N. Makhlouf ◽

Paul J. Logue ◽

Frederick H. Wilson ◽

Philip B. Dunham ◽

...

Keyword(s):

Functional Characterization ◽

Amino Acid Level ◽

Transmembrane Domains ◽

Chromosome 15 ◽

Hek 293 ◽

293 Cells ◽

Liver And Pancreas ◽

Large Extracellular Loop ◽

Hydropathy Analysis

We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75–76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and ∼20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH2 and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.

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Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]oregulation

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.5.c1516 ◽

1997 ◽

Vol 273 (5) ◽

pp. C1516-C1525 ◽

Cited By ~ 244

Author(s):

John A. Payne

Keyword(s):

Cell Lines ◽

Functional Characterization ◽

Cell Swelling ◽

Dual Function ◽

Kinetic Properties ◽

Hek 293 ◽

Apparent Affinity ◽

293 Cells ◽

The Brain

The neuronal K-Cl cotransporter isoform (KCC2) was functionally expressed in human embryonic kidney (HEK-293) cell lines. Two stably transfected HEK-293 cell lines were prepared: one expressing an epitope-tagged KCC2 (KCC2–22T) and another expressing the unaltered KCC2 (KCC2–9). The KCC2–22T cells produced a glycoprotein of ∼150 kDa that was absent from HEK-293 control cells. The 86Rb influx in both cell lines was significantly greater than untransfected control HEK-293 cells. The KCC2–9 cells displayed a constitutively active86Rb influx that could be increased further by 1 mM N-ethylmaleimide (NEM) but not by cell swelling. Both furosemide [inhibition constant ( K i) ∼25 μM] and bumetanide (Ki∼55 μM) inhibited the NEM-stimulated86Rb influx in the KCC2–9 cells. This diuretic-sensitive86Rb influx in the KCC2–9 cells, operationally defined as KCC2 mediated, required external Cl−but not external Na+ and exhibited a high apparent affinity for external Rb+(K+) [Michaelis constant ( K m) = 5.2 ± 0.9 (SE) mM; n = 5] but a low apparent affinity for external Cl−( K m >50 mM). On the basis of thermodynamic considerations as well as the unique kinetic properties of the KCC2 isoform, it is hypothesized that KCC2 may serve a dual function in neurons: 1) the maintenance of low intracellular Cl− concentration so as to allow Cl− influx via ligand-gated Cl− channels and 2) the buffering of external K+ concentration ([K+]o) in the brain.

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