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 (SG LT1) was inserted into the eukaryotic expression vector GFP-N1 under t he control of a CMV promoter. The plasmid was then stably transfected into a Chinese hamster ovary cell line (CHO). Transcription and synthe sis of SGLT1 were proved by Northern and Western blot analyses. Transp ort activities of the transfected cells (G6D3) were examined by measur ing the sodium-dependent uptake of alpha-methyl [C-14]D-glucoside (AMG ). Kinetic analysis revealed a V-max of 10.3 nmol/min/mg (total cell p rotein) and a K-m 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 mu M. Electrophysiological studies showe d that AMG induces a significant depolarization of membrane voltage in stably transfected CHO cells, suggesting an electrogenic Na-AMG sympo rt. Immunoprecipitation with an antipeptide antibody yielded a nearly homogeneous polypeptide with a molecular mass of about 72 kDa. The amo unt of SGLT1 present in the CHO cell plasma membranes represents at le ast 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 expressi on can serve as a promising model for studying cellular events related to Na+/D-glucose cotransport and for analyzing the structure and func tion of the cotransporter itself. (C) 1998 Elsevier Science B.V. All r ights reserved.