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.

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock

“…GUVs containing proteins, was a very challenging task. Several protocols have been proposed 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , mostly based on the ones established for protein-free GUVs. All these approaches have been successfully used with specific proteins.…”

Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model

“…These results demonstrated that the inhibitor driven MRP transporter inactivation results in increased mortality at least for two inhibitors, of the nematodes to Bcc strains, supporting the role of these transporters in Bcc infection. Verapamil has been characterized as a competitive inhibitor in ABCB1 malignant cell overexpression [ 52 ] as wells as a potent ABCC family inhibitor [ 53 ]. It is also involved in inhibiting C .…”

Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies

“…Hybrid lipid/polymer vesicles could be obtained via electroformation on Pt wires and ITO plates when using the lower voltage of lipids (3 V) . Both electroformation and gel‐assisted hydration showed that SUVs, in particular proteoliposomes, could be used as a substitute for the lipidic film to obtain proteoliposome GUVs with good reconstitution of transmembrane proteins …”

“…GPCRs are omnipresent in human cells, mediating extracellular signals for physiological signaling. Alternatively, proteoliposome GUVs can also be generated from the proteoliposome SUVs like for electroformation with replacing the lipid film with the SUVs . P‐glycoprotein, a member of transporters using ATP found in prokaryotes and eukaryotes, was successfully reconstituted in liposome GUVs.…”