Conformational changes in the intestinal brush border sodium-glucose cotransporter labeled with fluorescein isothiocyanate.

Abstract

Fluorescein isothiocyanate (FITC) was used to label the rabbit intestinal brush border Na+-glucose carrier, identify the carrier protein on sodium dodecyl sulfate/ polyacrylamide gel electrophoresis, and monitor the effect of ions and substrates on fluorescence quenching. Enriched brush border preparations were employed to study both glucose transport and FITC binding. FITC and a nonfluorescent analog (phenyl isothiocyanate, PITC) both inhibited Na+-dependent D-glucose transport irreversibly. Inhibition was blocked completely by the presence of Na+ and D-glucose during labeling. PITC was used to label nonspecific amino groups in the presence of glucose and Na+, and then the glucose carrier was labeled with FITC in the absence of substrates. Fluorescence of FITC bound to the carrier was quenched specifically with Na+ in a saturable fashion, and this indicates a Na+-dependent conformational change in the carrier. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of FITC-labeled membranes revealed specific labeling of a 71,000-dalton peptide. We conclude that Na+ induces a conformational shift in the 71,000-dalton glucose carrier, and this is quite consistent with the kinetics of Na+-dependent glucose transport in these membranes.Sodium-dependent organic solute transport across the brush border membrane of the small intestine plays a primary role in the absorption of sugars and amino acids (1). In these systems Na+ gradients provide the driving force for uphill solute uptake, and kinetic experiments suggest that Nat increases the affinity of the carrier for the solute through conformational changes. However, little direct information is available about either the nature of the conformation changes or even the identity of the carrier. Since site-specific reagents with fluorescent labels have been used successfully to probe conformation changes in other transport proteins (2-7), we have explored this approach with intestinal brush borders. Fluorescein isothiocyanate (FITC), an irreversible group-specific reagent for amino groups, has been used to label the Na+-glucose carrier, identify the carrier on NaDodSO4 gels, and monitor conformation changes. Nonspecific FITC binding was minimized by using enriched membrane preparations and labeling nonspecific sites with nonfluorescent isothiocyanates in the presence of excess Na+ and glucose. We find that Na+ produces a specific, saturable decrease in FITC fluorescence that is consistent with a Nat-dependent conformational change in the glucose camer. METHODSBrush border membrane vesicles (BBMVs) were isolated from rabbit small intestine by the Ca2t differential centrifugation method (8) and further purified by an adaptation of Hopfer's KSCN treatment (9). Briefly, the crude brush borders were resuspended by homogenization in 0.6 M KSCN and 10 mM Hepes/Tris buffer at pH 7.5, incubated at 40C, and then diluted 1:10 with 10 mM Hepes/Tris, pH 7.5-This suspension was centrifuged at 6000 x g for 10 min, and the resultant supernatant was centrifuged at 38,000 x g fo...

Histidyl residues at the active site of the Na/succinate cotransporter in rabbit renal brush borders

“…The Na/glucose cotransporter in rabbit small intestine seems to have a lysine residue at the glucose site (Weber & Semenza, 1983;Peerce & Wright, 1984) and possibly a tyrosine residue at the Na site in both kidney and intestine (Lin, Stroh & Kinne, 1982;B.E. Peerce, personal communication).…”

“…Evidence from Schmidt et at. (1983) and Peerce and Wright (1984) indicates that the Na/glucose transport protein has a molecular weight of 72,000 daltons. The H/monocarboxyl acid transporter in red blood cells, which is a possible cotransporter, may have a lysine/arginine group at the monocarboxyl site, since binding of DIDS can be specifically prevented by lactate.…”

The sodium/glucose cotransport family SLC5

“…This hypothesis was rapidly tested, refined and extended encompass the active transport of a diverse range of molecules and ions into virtually every cell type (see [64]). The first cotransporter proteins identified were the rabbit intestinal brush border Na + /glucose and Na + /proline cotransporters [55,79]. Stimulated by the success of others in cloning membrane proteins such as channels, pumps and transporters, we set out to clone cotransporters.…”

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

“…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.…”