Renal cortical basolateral Na⁺/HCO ⁻₃ cotransporter: II. Detection of conformational changes with fluorescein isothiocyanate labeling

Abstract

Fluorescein isothiocyanate (FITC) fluorescently labels amino groups and has been useful in detecting conformational changes in transport proteins through quenching or enhancement of the fluorescence signal upon exposure of protein to substrates. Solubilized renal basolateral membrane proteins, enriched in Na⁺/HCO ⁻₃ cotransporter activity, were reconstituted into liposomes and treated with FITC or its nonfluorescent analogue PITC (phenyl isothiocyanate). In the absence of Na⁺ and HCO ⁻₃ , incubation of proteoliposomes with PITC or FITC significantly inhibited cotransporter activity. However, in the presence of Na⁺ and HCO ⁻₃ during labeling both agents failed to inhibit cotransporter activity, indicating that these probes interact specifically with the cotransporter. In the presence of the substrates Na⁺ and HCO ⁻₃ , PITC binds covalently to amino groups unprotected by substrates leaving the Na⁺/HCO ⁻₃ cotransporter available for specific labeling with FITC. Addition of NaHCO₃ to FITC-labeled proteoliposomes resulted in a concentration-dependent enhancement of the fluorescence signal which was inhibited by pretreatment with 4,4′-diisothiocyanostilbene 2′,2-disulfonic acid (DIDS) prior to FITC labeling. SDS PAGE analysis of FITC-treated proteoliposomes showed the presence of two distinct fluorescent bands (approximate MW of 90 and 56 kD). In the presence of substrates, the fluorescence intensity of these bands was enhanced as confirmed by direct measurement of gel slice fluorescence. Thus, FITC detects conformational changes of the Na⁺/HCO ⁻₃ cotransporter and labels proteins which may represent the cotransporter or components of this cotransporter.

This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).