Protein kinase C phosphorylation disengages human and mouse-1a P-glycoproteins from influencing the rate of activation of swelling-activated chloride currents.
1. Whole-cell, swelling-activated Cl- currents, ICl(swell), were characterized in Chinese hamster ovary (CHO) cells and found to exhibit time-dependent inactivation at depolarizing potentials, tamoxifen and dideoxyforskolin sensitivity, and an anion permeability sequence: SCN- > I- > Br- > Cl- > F- > gluconate-. 2. CHO cells permanently transfected with either the human MDR1 or mouse mdr1a cDNAs demonstrated an increased rate of activation of ICl(swell) compared with parental cells or those permanently transfected with the mouse mdr1b cDNA. However, no differences in the magnitude of the currents were observed at steady state. 3. Pretreatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect ICl(swell) in MDR1 or mdr1a permanently transfected CHO cells. In contrast, pretreatment with TPA reduced ICl(swell) in MDR1(G185V)-expressing transfected NIH3T3 fibroblasts. Subsequently, the CHO cell lines were shown to contain significantly reduced levels of protein kinase C (PKC), suggesting that PKC concentrations might be limiting in these cell lines, at least under whole-cell patch clamp conditions. 4. Addition of purified PKC to the pipette solution, followed by a pretreatment with TPA, reduced the rate of ICl(swell) activation in human Pgp- and mouse Pgp1a-expressing CHO cells to the levels observed in parental and mouse Pgp1b-expressing cells. This confirms that PKC is limiting in these cells under whole-cell, patch clamp conditions. Furthermore, these results suggest that PKC-mediated phosphorylation of human Pgp and mouse Pgp1a disengages the influence which these Pgps have on ICl(swell). 5. These studies also demonstrate a functional distinction between the two mouse homologues, Pgp1a and Pgp1b. Although both can function as drug efflux pumps, only Pgp1a can act like human Pgp to influence ICl(swell).
Bond
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Nuffield Department of Clinical Biochemistry and Imperial Cancer Research Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Valverde
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MA
Higgins
C F
CF
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D048429:Cell Size; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D004594:Electrophysiology; D019450:Genes, MDR / Q000235:genetics / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018118:Chloride Channels; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate