ENHANCED LYSOSOMAL ACIDIFICATION LEADS TO INCREASED CHLOROQUINE ACCUMULATION IN CHO CELLS EXPRESSING THE PFMDR1 GENE

被引:54
作者
VANES, HHG
RENKEMA, H
AERTS, H
SCHURR, E
机构
[1] MCGILL UNIV,DEPT MED,MONTREAL,PQ,CANADA
[2] UNIV AMSTERDAM,EC SLATER INST BIOCHEM RES,1018 TV AMSTERDAM,NETHERLANDS
基金
英国医学研究理事会;
关键词
PLASMODIUM FALCIPARUM; CHLOROQUINE RESISTANCE; P-GLYCOPROTEIN; EXPRESSION SYSTEM;
D O I
10.1016/0166-6851(94)90166-X
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Expression of the pfmdr1-encoded Pgh1 protein of Plasmodium falciparum in CHO cells confers a phenotype of increased sensitivity to chloroquine due to an increased Pgh1-mediated accumulation of this antimalarial. Pgh1 carrying amino acid substitutions associated with chloroquine resistance in P. falciparum does not confer this phenotype. Here, we present studies on the underlying mechanism of Pgh1 mediated chloroquine influx into CHO cells. First, we measured intralysosomal pH using FITC-labelled dextran and found the intralysosomal pH in Pgh1 expressing cells to be decreased. A decreased lysosomal pH was not observed in cells expressing Pgh1 carrying the S1034C and N1042D double substitution found in some chloroquine-resistant P. falciparum parasites. Secondly, Pgh1-mediated uptake of chloroquine was abolished in the presence of bafilomycin A1, a specific inhibitor of vacuolar [H+]ATPases and was nearly abrogated in the presence of NH4Cl. Finally, cells expressing wild-type Pgh1 showed increased uptake of both (+)- and (-)[H-3]chloroquine enantiomers, indicating that Pgh1-mediated uptake of chloroquine is not enantioselective and in agreement with a pH-driven process. We conclude from these studies that Pgh1 does not transport chloroquine, but instead influences chloroquine accumulation by modulating the pH of acidic organelles. This function is abolished in Pgh1 carrying amino acid substitutions S1034C and N1042D. We speculate that the pfmdr1 gene encodes a vacuolar chloride channel.
引用
收藏
页码:209 / 219
页数:11
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