Volume-sensitive chloride channel activity does not depend on endogenous P-glycoprotein.
To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells.
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000096:biosynthesis* / Q000502:physiology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D017931:DNA Primers; D003630:Daunorubicin / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D004848:Epithelium; D006801:Humans; D007421:Intestine, Small; D008113:Liver Neoplasms; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D018118:Chloride Channels; D017931:DNA Primers; D004075:Diglycerides; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014750:Vincristine; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D003630:Daunorubicin