Molecular mechanism of two noncompetitive inhibitors of Na(+)-glucose cotransporter: comparison of DCCD and PCMB.
The effects of noncompetitive inhibitors of Na(+)-dependent glucose uptake, p-chloromercuribenzoate N,N'-dicyclohexylcarbodiimide (DCCD), on substrate-induced cotransporter conformational changes were examined using fluorescein isothiocyanate (FITC) and tryptophan fluorescence. p-chloromercuribenzoate (PCMB) inhibited both substrate-induced conformational changes with similar concentration required for 50% quenching/enhancement of tryptophan or FITC fluorescence. In contrast, DCCD inhibited the Na(+)-induced conformational change with an apparent concentration resulting in 50% inhibition (K0.5) of 18 microM and the glucose-induced conformational change with an apparent K0.5 of 5 microM. DCCD slightly increased the apparent K0.5 for the Na+ concentration required for Na(+)-induced conformational change. DCCD and PCMB altered tryptophan accessibility to quench reagents in all three conformations. Tryptophan residues on the PCMB-treated cotransporter were more Cs+ than I- sensitive in contrast to the unlabeled cotransporter. The PCMB-treated cotransporter had a reduced response to Na+, suggesting that the mode of PCMB inactivation of cotransporter activity resulted from conformational changes in the substrate-free cotransporter. DCCD had a smaller effect on cotransporter tryptophan quench reagent susceptibility. However, DCCD-labeled cotransporter was equally accessible to I- and Cs+, and the DCCD-labeled cotransporter did not respond to substrates. Loss of charge distribution around cotransporter tryptophans correlated with loss of substrate-induced conformational changes.
Peerce
B E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
D002729:Chloromercuribenzoates / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D008968:Molecular Conformation; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors*; D012964:Sodium / Q000494:pharmacology; D014364:Tryptophan / Q000502:physiology; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D009004:Monosaccharide Transport Proteins; D004024:Dicyclohexylcarbodiimide; D020245:p-Chloromercuribenzoic Acid; D014364:Tryptophan; D012964:Sodium; D016650:Fluorescein-5-isothiocyanate