Comparison of a Na+/D-glucose cotransporter from rat intestine expressed in oocytes of Xenopus laevis with the endogenous cotransporter.
Epithelial Na+/D-glucose cotransport was incorporated into the plasma membrane of Xenopus oocytes after microinjection of poly(A)(+)-mRNA from rat intestine tissue and was detected by measurements of uptake of [14C]AMG (methyl alpha-D-glucopyranoside). In mRNA-injected oocytes, the rate of AMG uptake exceeds the rate of endogenous Na+/AMG cotransport by a factor of up to 30. It is demonstrated that the additionally expressed transport differs qualitatively from the endogenous transport with respect to several parameters which is a prerequisite for the demonstration of expression of a foreign transporter: (1) The expressed system is more sensitive to external glucose or AMG and to the specific inhibitor phlorizin, (2) it is less sensitive to external Na+ and to changes in membrane potential, and (3) it is susceptible to inhibition by monoclonal antibodies, known to bind specifically to Na+/glucose cotransporters and to modulate the cotransport in kidney and intestine. The use of the antibodies allows one to distinguish between endogenous Na+/AMG cotransport and foreign cotransport expressed by injection of foreign mRNA. The expression of the foreign transport leads to transport rates that are high enough to detect the electrical current generated by the Na+/glucose cotransport. This allows future characterization of the cotransport system under voltage-clamp conditions by analyzing membrane current.
Weber
W M
WM
Max-Planck-Institut für Biophysik, Frankfurt/M, F.R.G.
Püschel
B
B
Steffgen
J
J
Koepsell
H
H
Schwarz
W
W
D000818:Animals; D000911:Antibodies, Monoclonal; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000378:metabolism; D007158:Immunologic Techniques; D007422:Intestines / Q000502:physiology*; D008564:Membrane Potentials; D008757:Methylglucosides / Q000378:metabolism; D008845:Microinjections; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D010695:Phlorhizin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000378:metabolism; D014982:Xenopus laevis
D000911:Antibodies, Monoclonal; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; C027020:methylglucoside; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose