Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts

We developed a confocal morphometric analysis to quantitate the relative pl asma membrane (PM) expression of the Na/H exchanger NHE3 in living PS120 fi broblasts. NHE3 is a membrane transport protein that is acutely regulated b y changes in the number of molecules expressed at the PM. To quantitate the PM expression of NHE3 under various experimental conditions, we stably exp ressed a chimera of rabbit NHE3 and green fluorescent protein (NHE3-GFP) in PS120 fibroblasts. A three-dimensional (3D) map of the intracellular distr ibution of NHE3-GFP was obtained by confocal laser scanning microscopy (CLS M) of cells superfused with a styryl dye, FM 4-64. This fluorophore rapidly and reversibly labeled the outer lipid layer of the PM, which allowed gene ration of a digital mask of the PM and calculation of the fraction of a tot al cellular NHE3-GFP expressed at the PM. This analysis was successfully us ed to quantitate the relative PM expression of NHE3-GFP in control cells (2 5%) and a decrease in the expression caused by subsequent exposure of cells to wortmannin (5.1%). Reliability of the method was confirmed by cell surf ace biotinylation, which yielded very similar results. Confocal morphometri c analysis is fast and reproducible and could potentially be used for inves tigations on regulation of expression of other membrane proteins.