Reconstitution of Human ABC Transporter Mrp3 into Giant Unilamellar Vesicles for Single Molecule Transport Recordings on Micro- Structured Biochips

Abstract

The functional reconstitution of large and complex membrane proteins such as eukaryotic ABC transporters into giant unilamellar liposomes (GUVs) represents a major challenge as GUV formation usually involves the presence of organic solvents and/or dehydration in high vacuum making it incompatible with delicate protein samples. To overcome this limitation, we developed a solvent-free method for the transformation of proteoliposomes into GUVs. MRP3-containing proteoliposomes were partly dehydrated on an agarose-based hydrogel under controlled humidity and in the presence of trehalose as a stabilizing agent. Subsequent rehydration in physiological buffer led to the fast and reproducible formation of GUVs (10-20 µm diameter) harboring functional MRP3 in their membrane.To observe the transport of substrates by single MRP3 molecules, MRP3-GUVs were fused onto the surface of a silicon-based biochip featuring a rectangular grid of thousands of cylindrical cavities (0.8 µm diameter, 6 fL volume) with open tops and optically transparent closed bottoms allowing highly parallel three-channel fluorescent readout on an inverted microscope set-up. Fluorescently labeled lipids in the bilayer and a fluorescent dye that is not transported by MRP3 served as in situ controls to continuously monitor the integrity of the pore-spanning lipid bilayer.ATP-dependent transport of autofluorescent substrates into the cavities by MRP3 could be monitored in real-time and revealed a distribution of rate constants in good agreement with previous bulk measurements. Furthermore, the competitive inhibition of MRP3-mediated transport by non-fluorescent co-substrates or inhibitors could also be observed.