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Functional expression of tagged human Na+-glucose cotransporter in Xenopus laevis oocytes.
Bissonnette P, Noël J, Coady MJ, Lapointe JY.
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1. High-affinity, secondary active transport of glucose in the intestine and kidney is mediated by an integral membrane protein named SGLT1 (sodium glucose cotransporter). Though basic properties of the transporter are now defined, many questions regarding the structure- function relationship of the protein, its biosynthesis and targeting remain unanswered. In order to better address these questions, we produced a functional hSGLT1 protein (from human) containing a reporter tag. 2. Six constructs, made from three tags (myc, haemaglutinin and poly-His) inserted at both the C- and N-terminal positions, were thus tested using the Xenopus oocyte expression system via electrophysiology and immunohistochemistry. Of these, only the hSGLT1 construct with the myc tag inserted at the N-terminal position proved to be of interest, all other constructs showing no or little transport activity. A systematic comparison of transport properties was therefore performed between the myc-tagged and the untagged hSGLT1 proteins. 3. On the basis of both steady-state (affinities for substrate (glucose) and inhibitor (phlorizin) as well as expression levels) and presteady-state parameters (transient currents) we conclude that the two proteins are functionally indistinguishable, at least under these criteria. Immunological detection confirmed the appropriate targeting of the tagged protein to the plasma membrane of the oocyte with the epitope located at the extracellular side. 4. The myc-tagged hSGLT1 was also successfully expressed in polarized MDCK cells. alpha-Methylglucose uptake studies on transfected cells showed an exclusively apical uptake pathway, thus indicating that the expressed protein was correctly targeted to the apical domain of the cell. 5. These comparative studies demonstrate that the myc epitope inserted at the N-terminus of hSGLT1 produces a fully functional protein while other epitopes of similar size inserted at either end of the protein inactivated the final protein.
Bissonnette,
Kinetic separation and characterization of three sugar transport modes in Caco-2 cells.
1996, Pubmed,
Xenbase
Bissonnette,
Kinetic separation and characterization of three sugar transport modes in Caco-2 cells.
1996,
Pubmed
,
Xenbase Bissonnette,
2-Deoxyglucose transport and metabolism in Caco-2 cells.
1996,
Pubmed Blais,
Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon.
1987,
Pubmed Chen,
Sodium leak pathway and substrate binding order in the Na+-glucose cotransporter.
1997,
Pubmed
,
Xenbase Chen,
Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter.
1996,
Pubmed
,
Xenbase Coady,
Functional studies of a chimeric protein containing portions of the Na(+)/glucose and Na(+)/myo-inositol cotransporters.
2000,
Pubmed
,
Xenbase DeWitt,
Targeting of two Arabidopsis H(+)-ATPase isoforms to the plasma membrane.
1996,
Pubmed Firsov,
Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.
1996,
Pubmed
,
Xenbase Geering,
A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes.
1989,
Pubmed
,
Xenbase Hediger,
Expression cloning and cDNA sequencing of the Na+/glucose co-transporter.
,
Pubmed
,
Xenbase Kong,
Targeting of recombinant Na+/glucose cotransporter (SGLT1) to the apical membrane.
1993,
Pubmed Lemas,
Assembly of Na,K-ATPase alpha-subunit isoforms with Na,K-ATPase beta-subunit isoforms and H,K-ATPase beta-subunit.
1994,
Pubmed Lostao,
Arginine-427 in the Na+/glucose cotransporter (SGLT1) is involved in trafficking to the plasma membrane.
1995,
Pubmed
,
Xenbase Martín,
Defects in Na+/glucose cotransporter (SGLT1) trafficking and function cause glucose-galactose malabsorption.
1996,
Pubmed
,
Xenbase Noel,
Differential localization of Na+/H+ exchanger isoforms (NHE1 and NHE3) in polarized epithelial cell lines.
1996,
Pubmed Panayotova-Heiermann,
Five transmembrane helices form the sugar pathway through the Na+/glucose cotransporter.
1997,
Pubmed
,
Xenbase Parent,
Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies.
1992,
Pubmed
,
Xenbase Robertson,
Ultrastructural localization of ras-related proteins using epitope-tagged plasmids.
1995,
Pubmed Sells,
Epitope-tag vectors for eukaryotic protein production.
1995,
Pubmed Smith,
Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.
1992,
Pubmed Suzuki,
Na(+)-dependent glucose transporter SGLT1 is localized in the apical plasma membrane upon completion of tight junction formation in MDCK cells.
1996,
Pubmed Turk,
Membrane topology of the human Na+/glucose cotransporter SGLT1.
1996,
Pubmed
,
Xenbase Turk,
Membrane topology motifs in the SGLT cotransporter family.
1997,
Pubmed Turner,
Carboxy-terminal vesicular stomatitis virus G protein-tagged intestinal Na+-dependent glucose cotransporter (SGLT1): maintenance of surface expression and global transport function with selective perturbation of transport kinetics and polarized expression.
1996,
Pubmed Vayro,
Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier.
1998,
Pubmed Yang,
Water and glycerol permeabilities of aquaporins 1-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes.
1997,
Pubmed
,
Xenbase
