CELL-VOLUME MEASURED BY TOTAL INTERNAL-REFLECTION MICROFLUORIMETRY - APPLICATION TO WATER AND SOLUTE TRANSPORT IN CELLS TRANSFECTED WITH WATER CHANNEL HOMOLOGS

Total internal reflection (TIR) microfluorimetry was established as a method to measure continuously the volume of adherent cells and applied to measure membrane permeabilities in cells transfected with water channel homologs. Cytosol was labeled with the membrane-impermeant fluorophore calcein. Fluorescence was excited by the TIR evanescent field in a thin section of cytosol (similar to 150 nm) adjacent to the cell-substrate interface. Because cytosolic fluorophore number per cell remains constant, the TIR fluorescence signal should be inversely related to cell volume. For small volume changes in Sf-9 and LLC-PK1 cells, relative TIR fluorescence was nearly equal to inverse relative cell volume; deviations from the ideal were modeled theoretically. To measure plasma membrane osmotic water permeability, P-f, the time course of osmotically induced cell volume change was inferred from the TIR fluorescence signal. LLC-PK1 cells expressing the CHIP28 water channel had an HgCl2- sensitive, threefold increase in P-f compared to nontransfected cells (P-f = 0.0043 cm/s at 10 degrees C). Solute permeability was measured from the TIR fluorescence time course in response to solute gradients. Glycerol permeability in Sf-9 cells expressing the water channel homolog GLIP was (1.3 +/- 0.2) x 10(-5).cm/s (22 degrees C), greater than that of (0.36 +/- 0.04) x 10(-5) cm/s (n = 4, p < 0.05) for control cells, indicating functional expression of GLIP. Water and urea permeabilities were similar in GLIP-expressing and control cells. The TIR method should be applicable to the study of water and solute permeabilities and cell volume regulation in cells of arbitrary shape and size.