Transfection of mu MDR 1 inhibits Na(+)-independent Cl(−)/(−)HCO(3) exchange in Chinese hamster ovary cells Journal Article

Authors: Luz, J. G.; Wei, L. Y.; Basu, S.; Roepe, P. D.
Article Title: Transfection of mu MDR 1 inhibits Na(+)-independent Cl(−)/(−)HCO(3) exchange in Chinese hamster ovary cells
Abstract: We have used single-cell photometry to measure intracellular pH (pHi) 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 (ΔΨ) 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 ΔΨ ≥ −40 (±5) mV and pHi ≤ 7.16 (±0.03) units. In contrast, MDR cell lines constructed by transfecting wild type mu MDR 1 cDNA are found to exhibit ΔΨ from 15 to 19 mV lower and pHi 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., & Talbot, F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7289–7293], exhibits elevated pHi and slightly elevated ΔΨ, 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., & Carlson, D. (1993) Biochemistry 32, 11042–11056] we find that overexpression of wild type mu MDR 1 protein inhibits Cl−- and −HCO3-dependent pHi homeostasis. Via single-cell photometry studies we now conclude that this is due to inhibition of Na+-independent C1−/−HCO3 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 ΔΨ and/or elevates pHi via CT−- and −HCO3-dependent mechanisms. © 1994, American Chemical Society. All rights reserved.
Keywords: controlled study; doxorubicin; nonhuman; mutant protein; animal cell; mouse; phenotype; animal; mice; gene expression; drug resistance; transfection; vinblastine; animalia; messenger rna; membrane glycoproteins; carrier proteins; murinae; immunoblotting; carboxyfluorescein; glutamic acid; sodium; phosphoprotein; multidrug resistance; hydrogen-ion concentration; complementary dna; p-glycoprotein; potassium; ribosome protein; colchicine; growth inhibition; cho cell; cho cells; cricetinae; cricetulus; cricetulus griseus; beta actin; membrane potentials; chlorides; bicarbonate; chloride ion; cell ph; cell membrane potential; cytophotometry; amiloride; dna transfection; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; antiporters; hamsters; anion exchange; valinomycin; bicarbonates; chloride-bicarbonate antiporters; 4 acetamido 4' isothiocyanatostilbene 2,2' disulfonic acid
Journal Title: Biochemistry
Volume: 33
Issue: 23
ISSN: 0006-2960
Publisher: American Chemical Society  
Date Published: 1994-06-14
Start Page: 7239
End Page: 7249
Language: English
DOI: 10.1021/bi00189a028
PROVIDER: scopus c2 - 7911682
PUBMED: 7911682
Notes: Export Date: 14 January 2019 -- Article -- Source: Scopus
Citation Impact
MSK Authors
  1. Subham   Basu
    7 Basu
  2. Paul D. Roepe
    26 Roepe