scholarly journals Mouse SGLT3a generates proton-activated currents but does not transport sugar

2012 ◽  
Vol 302 (8) ◽  
pp. C1073-C1082 ◽  
Author(s):  
Stephanie Barcelona ◽  
Danusa Menegaz ◽  
Ana Díez-Sampedro
Keyword(s):  
Mammalian Cells ◽  
Sugar Transport ◽  
Amino Acid Identity ◽  
Xenopus Laevis Oocytes ◽  
Acidic Ph ◽  
High Amino Acid ◽  
Inward Currents ◽  
Induced Currents ◽  
Dependent Transport ◽  
High Amino Acid Identity

Sodium-glucose cotransporters (SGLTs) are secondary active transporters belonging to the SLC5 gene family. SGLT1, a well-characterized member of this family, electrogenically transports glucose and galactose. Human SGLT3 (hSGLT3), despite sharing a high amino acid identity with human SGLT1 (hSGLT1), does not transport sugar, although functions as a sugar sensor. In contrast to humans, two different genes in mice and rats code for two different SGLT3 proteins, SGLT3a and SGLT3b. We previously cloned and characterized mouse SGLT3b (mSGLT3b) and showed that, while it does transport sugar like SGLT1, it likely functions as a physiological sugar sensor like hSGLT3. In this study, we cloned mouse SGLT3a (mSGLT3a) and characterized it by expressing it in Xenopus laevis oocytes and performing electrophysiology and sugar transport assays. mSGLT3a did not transport sugar, and sugars did not induce currents at pH 7.4, though acidic pH induced inward currents that increased in the presence of sugar. Moreover, mutation of residue 457 from glutamate to glutamine resulted in a Na+-dependent transport of sugar that was inhibited by phlorizin. To corroborate our results in oocytes, we expressed and characterized mSGLT3a in mammalian cells and confirmed our findings. In addition, we cloned, expressed, and characterized rat SGLT3a in oocytes and found characteristics similar to mSGLT3a. In summary, acidic pH induces currents in mSGLT3a, and sugar-induced currents are increased at acidic pH, but wild-type SGLT3a does not transport sugar.

Structure, function, and genomic organization of human Na+-dependent high-affinity dicarboxylate transporter

2000 ◽  
Vol 278 (5) ◽  
pp. C1019-C1030 ◽  
Author(s):  
Haiping Wang ◽  
You-Jun Fei ◽  
Ramesh Kekuda ◽  
Teresa L. Yang-Feng ◽  
Lawrence D. Devoe ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Catalytic Domain ◽  
Xenopus Laevis Oocytes ◽  
Ph Titration ◽  
High Affinity ◽  
Inward Currents ◽  
Induced Currents ◽  
Uptake Studies ◽  
A Charge ◽  
Carboxy Terminal

We have cloned and functionally characterized the human Na+-dependent high-affinity dicarboxylate transporter (hNaDC3) from placenta. The hNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the cloned transporter mediates the transport of succinate in the presence of Na+ [concentration of substrate necessary for half-maximal transport ( K t) for succinate = 20 ± 1 μM]. Dimethylsuccinate also interacts with hNaDC3. The Na+-to-succinate stoichiometry is 3:1 and concentration of Na+ necessary for half-maximal transport[Formula: see text]is 49 ± 1 mM as determined by uptake studies with radiolabeled succinate. When expressed in Xenopus laevis oocytes, hNaDC3 induces Na+-dependent inward currents in the presence of succinate and dimethylsuccinate. At a membrane potential of −50 mV,[Formula: see text] is 102 ± 20 μM and[Formula: see text]is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer and radiolabeled succinate uptake in hNaDC3-expressing oocytes indicate a charge-to-succinate ratio of 1:1 for the transport process, suggesting a Na+-to-succinate stoichiometry of 3:1. pH titration of citrate-induced currents shows that hNaDC3 accepts preferentially the divalent anionic form of citrate as a substrate. Li+inhibits succinate-induced currents in the presence of Na+. Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The human NaDC3 gene is located on chromosome 20q12–13.1, as evidenced by fluorescent in situ hybridization. The gene is >80 kbp long and consists of 13 exons and 12 introns.

scholarly journals Complete Genome Sequence of Murine Pneumotropic Virus (Polyomaviridae) Clone pKV(37-1)

2016 ◽  
Vol 4 (3) ◽  
Author(s):  
Jane E. Libbey ◽  
Robert S. Fujinami
Keyword(s):  
Regulatory Region ◽  
Viral Proteins ◽  
Amino Acid Identity ◽  
Virus Genome ◽  
T Antigens ◽  
Protein Coding ◽  
Coding Regions ◽  
High Amino Acid ◽  
Multiple Regions ◽  
High Amino Acid Identity

The murine pneumotropic virus genome encoded by the pKV(37-1) clone was sequenced to completion. The regulatory region harbored a mutation not previously reported. The protein coding regions (large and small T antigens, viral proteins 1 to 3) showed multiple regions of high amino acid identity to the human, simian, and bovine polyomaviruses.

scholarly journals Transport of butyryl-l-carnitine, a potential prodrug, via the carnitine transporter OCTN2 and the amino acid transporter ATB0,+

2007 ◽  
Vol 293 (5) ◽  
pp. G1046-G1053 ◽  
Author(s):  
Sonne R. Srinivas ◽  
Puttur D. Prasad ◽  
Nagavedi S. Umapathy ◽  
Vadivel Ganapathy ◽  
Prem S. Shekhawat
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
High Capacity ◽  
Amino Acid Transporter ◽  
Xenopus Laevis Oocytes ◽  
Loss Of Function ◽  
Bacterial Fermentation ◽  
Carnitine Transporter ◽  
Inward Currents ◽  
Acid Transporter

l-Carnitine is absorbed in the intestinal tract via the carnitine transporter OCTN2 and the amino acid transporter ATB0,+. Loss-of-function mutations in OCTN2 may be associated with inflammatory bowel disease (IBD), suggesting a role for carnitine in intestinal/colonic health. In contrast, ATB0,+ is upregulated in bowel inflammation. Butyrate, a bacterial fermentation product, is beneficial for prevention/treatment of ulcerative colitis. Butyryl-l-carnitine (BC), a butyrate ester of carnitine, may have potential for treatment of gut inflammation, since BC would supply both butyrate and carnitine. We examined the transport of BC via ATB0,+ to determine if this transporter could serve as a delivery system for BC. We also examined the transport of BC via OCTN2. Studies were done with cloned ATB0,+ and OCTN2 in heterologous expression systems. BC inhibited ATB0,+-mediated glycine transport in mammalian cells (IC50, 4.6 ± 0.7 mM). In Xenopus laevis oocytes expressing human ATB0,+, BC induced Na+-dependent inward currents under voltage-clamp conditions. The currents were saturable with a K0.5 of 1.4 ± 0.1 mM. Na+ activation kinetics of BC-induced currents suggested involvement of two Na+ per transport cycle. BC also inhibited OCTN2-mediated carnitine uptake (IC50, 1.5 ± 0.3 μM). Transport of BC via OCTN2 is electrogenic, as evidenced from BC-induced inward currents. These currents were Na+ dependent and saturable ( K0.5, 0.40 ± 0.02 μM). We conclude that ATB0,+ is a low-affinity/high-capacity transporter for BC, whereas OCTN2 is a high-affinity/low-capacity transporter. ATB0,+ may mediate intestinal absorption of BC when OCTN2 is defective.

scholarly journals WNT5B in Physiology and Disease

2021 ◽  
Vol 9 ◽  
Author(s):  
Sarocha Suthon ◽  
Rachel S. Perkins ◽  
Vitezslav Bryja ◽  
Gustavo A. Miranda-Carboni ◽  
Susan A. Krum
Keyword(s):  
Diabetes Mellitus ◽  
Cardiac Tissue ◽  
Expression Patterns ◽  
Cell Types ◽  
Amino Acid Identity ◽  
Acid Identity ◽  
High Amino Acid ◽  
Canonical Wnt ◽  
High Amino Acid Identity

WNT5B, a member of the WNT family of proteins that is closely related to WNT5A, is required for cell migration, cell proliferation, or cell differentiation in many cell types. WNT5B signals through the non-canonical β-catenin-independent signaling pathway and often functions as an antagonist of canonical WNT signaling. Although WNT5B has a high amino acid identity with WNT5A and is often assumed to have similar activities, WNT5B often exhibits unique expression patterns and functions. Here, we describe the distinct effects and mechanisms of WNT5B on development, bone, adipose tissue, cardiac tissue, the nervous system, the mammary gland, the lung and hematopoietic cells, compared to WNT5A. We also highlight aberrances in non-canonical WNT5B signaling contributing to diseases such as osteoarthritis, osteoporosis, obesity, type 2 diabetes mellitus, neuropathology, and chronic diseases associated with aging, as well as various cancers.

Structure, function, and regional distribution of the organic cation transporter OCT3 in the kidney

2000 ◽  
Vol 279 (3) ◽  
pp. F449-F458 ◽  
Author(s):  
Xiang Wu ◽  
Wei Huang ◽  
Malliga E. Ganapathy ◽  
Haiping Wang ◽  
Ramesh Kekuda ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Organic Cation ◽  
Regional Distribution ◽  
Human Kidney ◽  
Organic Cation Transporter ◽  
Mouse Kidney ◽  
Organic Cations ◽  
Xenopus Laevis Oocytes ◽  
Heterologous Expression Systems ◽  
Dependent Transport

We examined in this study the expression of the potential-sensitive organic cation transporter OCT3 in the kidney. A functionally active OCT3 was cloned from a mouse kidney cDNA library. The cloned transporter was found to be capable of mediating potential-dependent transport of a variety of organic cations including tetraethylammonium. This function was confirmed in two different heterologous expression systems involving mammalian cells and Xenopus laevis oocytes. We have also isolated the mouse OCT3 gene and deduced its structure and organization. The OCT3 gene consists of 11 exons and 10 introns. In situ hybridization studies in the mouse kidney have shown that OCT3 mRNA is expressed primarily in the cortex. The expression is evident in the proximal and distal convoluted tubules. The expression of OCT3 in human kidney was confirmed by RT-PCR. We have also cloned OCT3 from human placenta and human kidney. Human OCT3 exhibits 86% identity with mouse OCT3 in amino acid sequence. Human OCT3 was found to transport tetraethylammonium and a variety of other organic cations. The transport process was electrogenic. We conclude that OCT3 is expressed in mammalian kidney and that it plays an important role in the renal clearance of cationic drugs.

scholarly journals Melanocortin Receptor 4 (MC4R) Signaling System in Nile Tilapia

2020 ◽  
Vol 21 (19) ◽  
pp. 7036
Author(s):  
Tianqiang Liu ◽  
Yue Deng ◽  
Zheng Zhang ◽  
Baolong Cao ◽  
Jing Li ◽  
...  
Keyword(s):  
Amino Acid Identity ◽  
Melanocortin Receptor ◽  
Signaling System ◽  
Melanocyte Stimulating Hormone ◽  
High Amino Acid ◽  
Agonist And Antagonist ◽  
Agouti Related Protein ◽  
High Amino Acid Identity ◽  
Receptor Accessory Protein

The melanocortin receptor 4 (MC4R) signaling system consists of MC4R, MC4R ligands [melanocyte-stimulating hormone (MSH), adrenocorticotropin (ACTH), agouti-related protein (AgRP)], and melanocortin-2 receptor accessory protein 2 (MRAP2), and it has been proposed to play important roles in feeding and growth in vertebrates. However, the expression and functionality of this system have not been fully characterized in teleosts. Here, we cloned tilapia MC4R, MRAP2b, AgRPs (AgRP, AgRP2), and POMCs (POMCa1, POMCb) genes and characterized the interaction of tilapia MC4R with MRAP2b, AgRP, α-MSH, and ACTH in vitro. The results indicate the following. (1) Tilapia MC4R, MRAP2b, AgRPs, and POMCs share high amino acid identity with their mammalian counterparts. (2) Tilapia MRAP2b could interact with MC4R expressed in CHO cells, as demonstrated by Co-IP assay, and thus decrease MC4R constitutive activity and enhance its sensitivity to ACTH1-40. (3) As in mammals, AgRP can function as an inverse agonist and antagonist of MC4R, either in the presence or absence of MRAP2b. These data, together with the co-expression of MC4R, MRAP2b, AgRPs, and POMCs in tilapia hypothalamus, suggest that as in mammals, ACTH/α-MSH, AgRP, and MRAP2 can interact with MC4R to control energy balance and thus play conserved roles in the feeding and growth of teleosts.

scholarly journals The unprecedented diversity of UGT94-family UDP-glycosyltransferases in Panax plants and their contribution to ginsenoside biosynthesis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chengshuai Yang ◽  
Chaojing Li ◽  
Wei Wei ◽  
Yongjun Wei ◽  
Qunfang Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chemical Properties ◽  
Amino Acid Identity ◽  
Scientific Basis ◽  
Chain Elongation ◽  
Sugar Chain ◽  
Acid Identity ◽  
Catalytic Function ◽  
High Amino Acid ◽  
High Amino Acid Identity

Abstract More than 150 ginsenosides have been isolated and identified from Panax plants. Ginsenosides with different glycosylation degrees have demonstrated different chemical properties and bioactivity. In this study, we systematically cloned and characterized 46 UGT94 family UDP-glycosyltransferases (UGT94s) from a mixed Panax ginseng/callus cDNA sample with high amino acid identity. These UGT94s were found to catalyze sugar chain elongation at C3-O-Glc and/or C20-O-Glc of protopanaxadiol (PPD)-type, C20-O-Glc or C6-O-Glc of protopanaxatriol (PPT)-type or both C3-O-Glc of PPD-type and C6-O-Glc of PPT-type or C20-O-Glc of PPD-type and PPT-type ginsenosides with different efficiencies. We also cloned 26 and 51 UGT94s from individual P. ginseng and P. notoginseng plants, respectively; our characterization results suggest that there is a group of UGT94s with high amino acid identity but diverse functions or catalyzing activities even within individual plants. These UGT94s were classified into three clades of the phylogenetic tree and consistent with their catalytic function. Based on these UGT94s, we elucidated the biosynthetic pathway of a group of ginsenosides. Our present results reveal a series of UGTs involved in second sugar chain elongation of saponins in Panax plants, and provide a scientific basis for understanding the diverse evolution mechanisms of UGT94s among plants.

Kinetic separation and characterization of three sugar transport modes in Caco-2 cells

1996 ◽  
Vol 270 (5) ◽  
pp. G833-G843 ◽  
Author(s):  
P. Bissonnette ◽  
H. Gagne ◽  
M. J. Coady ◽  
K. Benabdallah ◽  
J. Y. Lapointe ◽  
...  
Keyword(s):  
Cell Line ◽  
Choline Chloride ◽  
Sugar Transport ◽  
Salient Feature ◽  
Mixed System ◽  
Xenopus Laevis Oocytes ◽  
Chloride Uptake ◽  
System A ◽  
Dependent Transport ◽  
Colonic Cells

The question of sugar transport heterogeneity in the human intestinal Caco-2 cell line was addressed using alpha-methyl-D-glucose (AMG) and 2-deoxy-D-glucose (DG) as substrate analogues for D-glucose, the transport inhibitors phlorizin (PZ) and phloretin (PT), and NaCl or choline chloride uptake media. The data are compatible with the existence of three distinct pathways that can be isolated kinetically according to specific characteristics: 1) an “:AMG-strict” system, strictly Na+ dependent and specific for AMG [Michaelis-Menten constant value (K(m)) = 2.0 +/- 0.3 mM] but sensitive to both PZ and PT, with PZ being more potent than PT, 2) a “DG-strict” system, strictly Na+ independent and specific for both DG (K(m) = 5.2 +/- 0.5 mM) and PT; and 3) a “DG/AMG-mixed” system, strictly Na+ dependent, with loose specificities for the glucose analogues DG (K(m) = 0.81 +/- 0.07 mM) and AMG (K(m) = 8.1 +/- 0.8 mM), and the inhibitors PZ and PT, but with PT being more potent than PZ. Since SGLT-1 obtained by polymerase chain reaction from either Caco-2 cells or normal human jejunum demonstrated identical transport properties when expressed in Xenopus laevis oocytes, we conclude that the “AMG-strict” system represents the expression of human SGLT-1 activity in this cell line. Moreover, Western blot analysis revealed that SGLT-1 is located exclusively in the apical membrane. In contrast, neither the nature nor the membrane location of both the DG-strict and DG/AMG-mixed pathways could be resolved unambiguously. Still it has been demonstrated that expression of the latter system is constitutive to all Caco-2 cells and that its Na+ dependence is not the consequence of H(+)-dependent transport activity. Aside from the presence of the DG/AMG-mixed system, a salient feature of Caco-2 cells is that the GLUT-3 protein is located exclusively in the brush-border membrane. Due to these limitations, it is concluded that the Caco-2 cell line cannot be considered as equivalent to either fetal colonic cells or normal enterocytes.

scholarly journals Cloning of a Gene Encoding Raw-Starch-Digesting Amylase from a Cytophaga sp. and Its Expression in Escherichia coli

2002 ◽  
Vol 68 (7) ◽  
pp. 3651-3654 ◽  
Author(s):  
Chii-Ling Jeang ◽  
Li-Shien Chen ◽  
Ming-Yu Chen ◽  
Rong-Jen Shiau
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid Identity ◽  
Protein Product ◽  
Gene Encoding ◽  
Raw Starch ◽  
Acid Identity ◽  
Expression In Escherichia Coli ◽  
High Amino Acid ◽  
High Amino Acid Identity

ABSTRACT A raw-starch-digesting amylase (RSDA) gene from a Cytophaga sp. was cloned and sequenced. The predicted protein product contained 519 amino acids and had high amino acid identity to α-amylases from three Bacillus species. Only one of the Bacillus α-amylases has raw-starch-digesting capability, however. The RSDA, expressed in Escherichia coli, had properties similar to those of the enzyme purified from the Cytophaga sp.

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