NOVEL CL--DEPENDENT INTRACELLULAR PH REGULATION IN MURINE MDR-1 TRANSFECTANTS AND POTENTIAL IMPLICATIONS

Previously [Luz et al. (1994) Biochemistry 33, 7239-7249], we determined that Cl-- and (HCO3)-H---dependent pH(i) homeostasis was perturbed in multidrug resistant (MDR) cells created by transfecting LR73 Chinese hamster ovary fibroblasts with wild-type mu (murine) MDR 1 (Gros et al., 1991). Via single-cell photometry experiments performed under various conditions, we are now able to separate Na+-dependent and Na+-independent components of Cl-/(HCO3)-H-- exchange in the MDR transfectants and the parental LR73 cells. Cl--dependent, Na+-independent reacidification of pH(i), mediated by the anion exchanger 2 isoform in LR73 cells, is dramatically inhibited by mild overexpression of MDR protein. Analysis of H+ flux at different pH(i) shows that Cl--dependent reacidification approaches 0.2 mM H+/s for LR73 cells at pH(i) = 8.0 but is at least 10-fold slower for MDR 1 transfectants that were never exposed to chemotherapeutics (EX4N7 cells). MDR 1 transfectants selected on the chemotherapeutic vinblastine (1-1 cells), which express approximately 10-fold more MDR protein relative to EX4N7 cells, exhibit similar behavior; however, alterations in Cl--dependent pH(i) regulation are more severe. Hypotonic conditions, which have been shown to increase anomalous Cl- conductance in some cells overexpressing MDR protein (Valverde et al., 1992), are found to amplify the altered pH(i) homeostasis features in the primary transfectants that express lower levels of MDR protein such that they then mimic the behavior of the drug-selected cells that express substantially more MDR protein. Verapamil reverses the anomalous behavior. In addition, removal of CO2 causes similar pH(i) changes in all cell types, whereas removal of (HCO3)-H-- in the presence of Cl- leads to greater changes in pH(i) for the MDR cells. Transfectants harboring mutant MDR protein that is unable to confer the MDR phenotype (K432R/K1074R mu MDR 1) perform Na+-independent Cl-/(HCO3)-H-- exchange similar to the parental LR73 cells. These data may help to further refine models for MDR protein function in MDR cells.