Examination of the sodium-induced conformational change of the intestinal brush border sodium/glucose symporter using fluorescent probes

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Abstract
The Na+-induced change in conformation of the intestinal brush border glucose carrier has been examined by three procedures. In the first, we have measured the effect of Na+ on the binding of fluorescein isothiocyanate (FITC) to the glucose site; 100 mM Na increased the specific [blocked by D-glucose, p-(chloromercuri)benzenesulfonic acid, and N-acetylimidazole] FITC binding to a 75-kilodalton polypeptide 3-fold. In the second series, we have examined the effect of Na+ on the susceptibility of the fluorescently labeled glucose site [pyrene isothiocyanate (PYTC) labeled] to a hydrophilic quencher (Tl+); 100 mM NaCl increased the fraction of PYTC sites available to Tl+ from 32% to 92% and decreased the apparent quenching constant from 94 to 44 M-1. Finally, in the third series, we probed the distribution of tryptophan residues 15-30 .ANG. from the glucose site using a "distant reporter group method", where tryptophan was used as an energy donor to anthracene isothiocyanate bound to the glucose site. Tryptophan quench reagents (I-, Cs+, and acrylamide) were then employed to probe the accessibility of the glucose site tryptophans in the presence and absence of sodium. In the absence of Na+, there were two major classes of glucose tryptophans.sbd.exterior surface residues and residues buried in the hydrophobic protein matrix. Na+ caused a redistribution of the donor tryptophans such that a higher percentage were accessible to I- (51% vs. 25%) and fewer were accessible to Cs+ (13% vs. 25%) and acrylamide (27% vs. 57%). These results indicate that during the Na+-induced conformational change, there is a redistribution of tryptophans at the surface of the protein. These short- and long-range conformational changes induced by Na+ are consistent with the Na+-induced increase in accessibility of the glucose site to D-glucose, phlorizin, and FITC.
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