title: Purified human MDR 1 modulates membrane potential in reconstituted proteoliposomes.
abstract: Human multidrug resistance (hu MDR 1) cDNA was fused to a P. shermanii transcarboxylase biotin acceptor domain (TCBD), and the fusion protein was heterologously overexpressed at high yield in K(+)-uptake deficient Saccharomyces cerevisiae yeast strain 9.3, purified by avidin-biotin chromatography, and reconstituted into proteoliposomes (PLs) formed with Escherichia coli lipid. As measured by pH- dependent ATPase activity, purified, reconstituted, biotinylated MDR-TCBD protein is fully functional. Dodecyl maltoside proved to be the most effective detergent for the membrane solubilization of MDR-TCBD, and various salts were found to significantly affect reconstitution into PLs. After extensive analysis, we find that purified reconstituted MDR-TCBD protein does not catalyze measurable H(+) pumping in the presence of ATP. In the presence of physiologic [ATP], K(+)/Na(+) diffusion potentials monitored by either anionic oxonol or cationic carbocyanine are easily established upon addition of valinomycin to either control or MDR-TCBD PLs. However, in the absence of ATP, although control PLs still maintain easily measurable K(+)/Na(+) diffusion potentials upon addition of valinomycin, MDR-TCBD PLs do not. Dissipation of potential by MDR-TCBD is clearly [ATP] dependent and also appears to be Cl(-) dependent, since replacing Cl(-) with equimolar glutamate restores the ability of MDR-TCBD PLs to form a membrane potential in the absence of physiologic [ATP]. The data are difficult to reconcile with models that might propose ATP-catalyzed "pumping" of the fluorescent probes we use and are more consistent with electrically passive anion transport via MDR-TCBD protein, but only at low [ATP]. These observations may help to resolve the confusing array of data related to putative ion transport by hu MDR 1 protein.
authors.affiliation: Department of Chemistry, Lombardi Cancer Center Program in Tumor Biology, Georgetown University, 37th and O Streets, Washington, DC 20057, USA.
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Howard
authors.forename: Paul D
authors.initials: PD
authors.lastname: Roepe
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D017136:Ion Transport; D007555:Isoxazoles; D008564:Membrane Potentials; D011424:Propionibacterium / Q000737:chemistry / Q000235:genetics; D011510:Proteolipids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D007555:Isoxazoles; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; C020485:proteoliposomes; C013633:oxonol V; D000255:Adenosine Triphosphate; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D000251:Adenosine Triphosphatases