TRANSFECTION OF MU-MDR-1 INHIBITS NA+-INDEPENDENT CL-/(HCO3)-H-- EXCHANGE IN CHINESE-HAMSTER OVARY CELLS

We have used single-cell photometry to measure intracellular pH (pH(i)) for several MDR cell lines constructed by stably transfecting LR73 chinese hamster ovary fibroblasts with mutant and wild type murine MDR 1 genes. In addition, plasma membrane electrical potential (Delta Psi) has been measured for the same cells by the K+/valinomycin null point titration method using the ratiometric styryl probe di-4-ANEPPS. Both the untransfected, parental cell line and a cell line expressing substantial mutant MDR 1 protein (K432R/K1074R) that is unable to confer the MDR phenotype are found to have Delta Psi greater than or equal to -40 (+5) mV and pH(i) less than or equal to 7.16 (+0.03) units. In contrast, MDR cell lines constructed by transfecting wild type mu MDR 1 cDNA are found to exhibit Delta Psi from 15 to 19 mV lower and pH(i) from 0.13 to 0.34 units higher. A cell line that overexpresses crippled MDR protein (S941F) that is not resistant to colchicine or doxorubicin, but which is resistant to vinblastine [Gros, P., Dhir, R., Croop, J., and Talbot, F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7289-7293], exhibits elevated pH(i) and slightly elevated Delta Psi, relative to LR73. Northern and western blot analyses confirm the substantial overexpression of the mu MDR genes and proteins in these lines, as well as the mild overexpression of endogenous hamster p-GP mRNA in some lines. In general agreement with previous studies that examined myeloma cells overexpressing hu MDR 1 protein [Roepe, P. D., Wei, L.-Y., Cruz, J., and Carlson, D. (1993) Biochemistry 32, 11042-11056] we find that overexpression of wild type mu MDR 1 protein inhibits Cl-- and (HCO3)-H---dependent pH(i) homeostasis. Via, single-cell photometry studies we now conclude that this is due to inhibition of Na+-independent Cl-/(HCO3)-H-- exchange (strict anion exchange or AE). As concluded previously for other MDR cells, decreased AE activity is not due to decreased expression of the exchanger; in fact, again similar to previous work [Roepe et al. (1993) Biochemistry 32, 11042-11056], we find increased levels of AE mRNA in some MDR cell lines. Models that may explain these data that are also consistent with the known physiology of cells that endogenously express MDR protein are suggested. These data are consistent with a model for MDR protein function wherein overexpression of the protein decreases Delta Psi and/or elevates pH(i) via Cl-- and (HCO3)-H---dependent mechanisms.