Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells.
Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.
Birnir
B
B
Department of Physiology, UCLA School of Medicine, 90024-1751.
Lee
H S
HS
Hediger
M A
MA
Wright
E M
EM
D000818:Animals; D050260:Carbohydrate Metabolism; D002460:Cell Line; D015870:Gene Expression; D006031:Glycosylation; D000882:Haplorhini; D007413:Intestinal Mucosa / Q000378:metabolism*; D007477:Ions; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D010957:Plasmids; D014162:Transfection
D007477:Ions; D009004:Monosaccharide Transport Proteins