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.

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.

The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)I RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl a-D-[1"Clglucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (V.)of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/ glucose cotransporter was found in a single fraction of "z2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.Active glucose absorption across the small intestine is performed by enterocytes at the tip of the villus. Transport occurs in two stages: the first is intracellular accumulation of the sugar across the brush-border membrane by a sodium/ glucose cotransporter, and the second is facilitated diffusion of sugar out of the cell across the basolateral membrane.Recently, the sodium/glucose cotransporter has been identified as a 75-kDa polypeptide (1, 2), and some progress has been made in the characterization of this transport protein (3). The facilitated glucose carrier in the basolateral membrane is probably similar, if not identical, to the 55-kDa glucose carrier in human erythrocytes (see refs. 4-6).Intestinal glucose absorption exhibits adaptive regulation with diet, starvation, diabetes, gestation, lactation, and aging (7). For example, in diabetes mellitus, the maximal velocity (Vmax) of sodium-dependent glucose transport increases, and this is probably due to an increase in the number of sodium/glucose cotransporters. There is also a genetic component, and this is best exemplified by the rare hereditary disorder called primary glucose/galactose malabsorption (see ref. 8), which again is probably due to a defect in the cotransporter.As a first step in cloning the gene for the intestinal sodium/glucose cotransporter, we have attempted to express the carrier in Xenopus laevis oocytes and to identify the size of the mRNA encoding the 75-kDa protein. Amphibian oocytes translate foreign mRNAs very efficiently (9) and have been successfully exploited to express functional membrane transport proteins ranging from ion channels to ion exchangers (10-12). Here we demonstrate that Xenopus oocytes successfully translate i...

Injection of Xenopus laevis oocytes with rabbit heart poly(A)+RNA results in expression of Na+ inside (Nai+)-dependent Ca2+ uptake activity. The activity was measured by first loading the oocytes with Na+ using nystatin and then incubating the oocytes in K+ or Na+ medium containing 45Ca. The expressed Na+ gradient-dependent Ca2+ uptake was five to eight times that observed with water-injected oocytes or with poly(A)+RNA-injected oocytes for which the Na+ load step had been omitted. Induced activity was related to the amount of RNA injected and was insensitive to nifedipine. Fractionation of the poly(A)+RNA on a sucrose gradient determined that the active message had a size range between 3 and 5 kb. The properties of the Na+ gradient-dependent Ca2+ uptake indicated that Na+-Ca2+ exchange activity had been expressed in X. laevis oocytes. The result may be useful for cloning and identifying the molecular component responsible for Na+-Ca2+ exchange.