title: Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes.
abstract: This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (Ps) of passive diffusion and rate constants of active transport (kT) by P-gp as a result of different experimental conditions. The Ps value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the kT value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in kT values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.
authors.affiliation: Department of Chemical Engineering, University of Michigan, 2300 Hayward Street, Ann Arbor, MI 48109, USA.
authors.forename: Kim S
authors.initials: KS
authors.lastname: Horger
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Avenue, Ann Arbor, MI 48109, USA.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Avenue, Ann Arbor, MI 48109, USA.
authors.forename: Divya K
authors.initials: DK
authors.lastname: Rao
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20814, USA.
authors.forename: Suneet
authors.initials: S
authors.lastname: Shukla
authors.affiliation: Department of Biomedical Engineering, University of Michigan, 1101 Beal Avenue, Ann Arbor, MI 48109, USA; Center for Computational Medicine and Bioinformatics, University of Michigan, 2800 Plymouth Road, Ann Arbor, MI 48109, USA.
authors.forename: David
authors.initials: D
authors.lastname: Sept
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20814, USA.
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.affiliation: Department of Chemical Engineering, University of Michigan, 2300 Hayward Street, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, University of Michigan, 1101 Beal Avenue, Ann Arbor, MI 48109, USA. Electronic address: mimayer@umich.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Mayer
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D004058:Diffusion; D005456:Fluorescent Dyes; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D007700:Kinetics; D007915:Lepidoptera / Q000737:chemistry; D015233:Models, Statistical; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D011510:Proteolipids / Q000737:chemistry* / Q000648:ultrastructure; D020112:Rhodamine 123; D012685:Sepharose / Q000737:chemistry; D019076:Transgenes; D014700:Verapamil / Q000494:pharmacology
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D005456:Fluorescent Dyes; D020100:Hydrogels; D011510:Proteolipids; C020485:proteoliposomes; D020112:Rhodamine 123; D000255:Adenosine Triphosphate; D012685:Sepharose; D014700:Verapamil
keywords: Chloride ion channel; Liposome; P-glycoprotein; Permeability; Proteoliposome; Transport rate constant