Demonstration of sodium-dependent,electrogenic substrate transport in rat small intestinal brush border membrane vesicles by a cyanine dye

The cyanine dye DiS-C₂(5) was tested as an indicator for changes in membrane potential of subfractionated rat jejunal brush border membrane vesicles. The fluorescence of this dye increased with inside positive and decreased with inside negative potentials. The sensitivity to inside negative potentials was greater than to inside positive potentials. The addition ofl-alanine,l-phenylalanine,l-methionine,d-galactose andd-glucose in the presence of sodium provoked a transient fluorescence increase indicating an inside positive membrane potential due to electrogenic, sodium-coupled transport. Besides the sodium-dependence, the dye reflected stereo-specificity and saturability ofd-glucose transport. Whend-glucose loaded vesicles were incubated ind-glucose-free medium, a decrease in fluorescence was observed indicating thatd-glucose efflux is also electrogenic.Abbreviations Dis-C₂(5) 3,3-diethylthiadicarbocyanine iodide - DiS-C₃(5) 3,3-dipropylthiadicarbocyanine iodide - HEPES N-2-hydroxyethylpiperazine-N-ethane sulfonic acid - Tris tris(hydroxymethyl)aminomethane - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N-tetraacetic acid