ABSTRACT
The Na+/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed in Xenopus oocytes. Injection of cloned RNA into oocytes increased Na+/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed in Xenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of 14C-alpha-methyl-D-glucopyranoside uptake into brush border membrane vesicles and Xenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na+-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (Ki 6 and 9 microM), and saturable (alpha-methyl-D-glucopyranoside Km 110 and 570 microM). The sugar specificity was examined by competition experiments, and in both cases the selectivity was D-glucose greater than alpha-methyl-D-glucopyranoside greater than D-galactose greater than 3-O-methyl-D-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na+/glucose cotransporter.
Subject(s)
Intestine, Small/analysis , Monosaccharide Transport Proteins/analysis , Animals , Cloning, Molecular , Glucose/metabolism , Glucose/pharmacokinetics , Intestine, Small/metabolism , Intestine, Small/physiology , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Phlorhizin/pharmacology , Rabbits , Sodium/metabolism , Sodium/pharmacokinetics , Substrate SpecificityABSTRACT
Organic substrates (sugars, amino acids, carboxylic acids and neutrotransmitters) are actively transported into eukaryotic cells by Na+ co-transport. Some of the transport proteins have been identified--for example, intestinal brush border Na+/glucose and Na+/proline transporters and the brain Na+/CI-/GABA transporter--and progress has been made in locating their active sites and probing their conformational states. The archetypical Na+-driven transporter is the intestinal brush border Na+/glucose co-transporter (see ref. 8), and a defect in the co-transporter is the origin of the congenital glucose-galactose malabsorption syndrome. Here we describe cloning of this co-transporter by a method new to membrane proteins. We have sequenced the cloned DNA and have found no homology between the Na+/glucose co-transporter and either the mammalian facilitated glucose carrier or the bacterial sugar transport proteins. This suggests that the mammalian Na+-driven transporter has no evolutionary relationship to the other sugar transporters.
