High-level expression of Na+/D-glucose cotransporter (SGLT1) in a stably transfected Chinese hamster ovary cell line.
The coding region of the high affinity Na+/d-glucose cotransporter (SGLT1) was inserted into the eukaryotic expression vector GFP-N1 under the control of a CMV promoter. The plasmid was then stably transfected into a Chinese hamster ovary cell line (CHO). Transcription and synthesis of SGLT1 were proved by Northern and Western blot analyses. Transport activities of the transfected cells (G6D3) were examined by measuring the sodium-dependent uptake of alpha-methyl[14C]d-glucoside (AMG). Kinetic analysis revealed a Vmax of 10.3 nmol/min/mg (total cell protein) and a Km of 0.26+/-0.09 mM, respectively. The concentration of phlorizin required to inhibit AMG uptake by 50% in the presence of 0.1 mM AMG was 2.35+/-1.84 microM. Electrophysiological studies showed that AMG induces a significant depolarization of membrane voltage in stably transfected CHO cells, suggesting an electrogenic Na-AMG symport. Immunoprecipitation with an antipeptide antibody yielded a nearly homogeneous polypeptide with a molecular mass of about 72 kDa. The amount of SGLT1 present in the CHO cell plasma membranes represents at least 1% of membrane protein, which is about 30-100 times higher than in natural sources, such as renal brush border membranes. In conclusion, the stably transfected G6D3 cells with a markedly high SGLT1 expression can serve as a promising model for studying cellular events related to Na+/d-glucose cotransport and for analyzing the structure and function of the cotransporter itself.
Lin
J T
JT
Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Rheinlanddamm 201, 44139 Dortmund, Germany.
Kormanec
J
J
Wehner
F
F
Wielert-Badt
S
S
Kinne
R K
RK
D000818:Animals; D001692:Biological Transport / Q000235:genetics; D019921:Biotinylation; D016466:CHO Cells; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics; D004594:Electrophysiology; D005786:Gene Expression Regulation / Q000235:genetics*; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008757:Methylglucosides / Q000493:pharmacokinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010695:Phlorhizin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014162:Transfection / Q000235:genetics
D008562:Membrane Glycoproteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D051269:Sodium-Glucose Transporter 1; C027020:methylglucoside; D010695:Phlorhizin