RENAL CORTICAL BASOLATERAL NA+ HCO3- COTRANSPORTER .2. DETECTION OF CONFORMATIONAL-CHANGES WITH FLUORESCEIN ISOTHIOCYANATE LABELING/

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups an d has been useful in detecting conformational changes in transport pro teins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral memb rane proteins, enriched in Na+/HCO3- cotransporter activity, were reco nstituted into liposomes and treated with FITC or its nonfluorescent a nalogue PITC (phenyl isothiocyanate). In the absence of Na+ and HCO3-, incubation of proteoliposomes with PITC or FITC significantly inhibit ed cotransporter activity. However, in the presence of Na+ and HCO3- d uring labeling both agents failed to inhibit cotransporter activity, i ndicating that these probes interact specifically with the cotransport er. In the presence of the substrates Na+ and HCO3-, PITC binds covale ntly to amino groups unprotected by substrates leaving the Na+/HCO3- c otransporter available for specific labeling with FITC. Addition of Na HCO3 to FITC-labeled proteoliposomes resulted in a concentration-depen dent enhancement of the fluorescence signal which was inhibited by pre treatment with 4,4'-diisothiocyanostilbene 2',2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteolipos omes showed the presence of two distinct fluorescent bands (approximat e MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurem ent of gel slice fluorescence. Thus, FITC detects conformational chang es of the Na+/HCO3- cotransporter and labels proteins which may repres ent the cotransporter or components of this cotransporter.