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Quantitation of Plasma Membrane Expression of a Fusion Protein of Na/H Exchanger NHE3 and Green Fluorescence Protein (GFP) in Living PS120 Fibroblasts
Andrzej J. Janeckia, Maria Janeckib, Shafinaz Akhterc, and Mark Donowitzca The University of Texas–Medical School at Houston, Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, Houston, Texas
b Baylor College of Medicine, Department of Cell Biology, Houston, Texas
c The Johns Hopkins University School of Medicine, Departments of Medicine and Physiology, Division of Gastroenterology, Baltimore, Maryland
Correspondence to: Andrzej J. Janecki, Div. of Gastroenterology, Hepatology and Nutrition, U. of Texas Medical School at Houston, 6431 Fannin, 4.234 MSB, Houston, TX 77030. E-mail: ajaneck@heart.med.uth.tmc.edu
We developed a confocal morphometric analysis to quantitate the relative plasma membrane (PM) expression of the Na/H exchanger NHE3 in living PS120 fibroblasts. NHE3 is a membrane transport protein that is acutely regulated by changes in the number of molecules expressed at the PM. To quantitate the PM expression of NHE3 under various experimental conditions, we stably expressed a chimera of rabbit NHE3 and green fluorescent protein (NHE3–GFP) in PS120 fibroblasts. A three-dimensional (3D) map of the intracellular distribution of NHE3–GFP was obtained by confocal laser scanning microscopy (CLSM) 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 generation of a digital mask of the PM and calculation of the fraction of a total cellular NHE3–GFP expressed at the PM. This analysis was successfully used to quantitate the relative PM expression of NHE3–GFP in control cells (25%) and a decrease in the expression caused by subsequent exposure of cells to wortmannin (5.1%). Reliability of the method was confirmed by cell surface biotinylation, which yielded very similar results. Confocal morphometric analysis is fast and reproducible and could potentially be used for investigations on regulation of expression of other membrane proteins. (J Histochem Cytochem 48:1479–1491, 2000)
Key Words: NHE3, confocal microscopy, green fluorescent protein, morphometric analysis
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