Microfluidic-Derived Detection of Protein-Facilitated Copper Flux Across Lipid Membranes

Abstract

Measurement of protein-facilitated copper flux across biological membranes is a considerable challenge. Here, we demonstrate a straightforward microfluidic-derived approach for visualization and measurement of membranous Cu flux. Giant unilamellar vesicles, reconstituted with the membrane protein of interest, are prepared, surface-immobilized, and assessed using a novel quencher-sensor reporter system for detection of copper. With the aid of a syringe pump, the external buffer is exchanged, enabling consistent and precise exchange of solutes, without causing vesicle rupture or uneven local metal concentrations brought about by rapid mixing. This approach bypasses common issues encountered when studying heavy metal-ion flux, thereby providing a new platform for in vitro studies of metal homeostasis aspects that are critical for all cells, health, and disease.

Figure 1

Schematic of the microfluidic setup for measurement of Cu flux in giant unilamellar vesicles. The three magnifications of the experiment are shown: the equipment used for the experiments, the process observed in the channel slides, and the molecular mechanism of the reactions.

Figure 2

Micrographs of representative samples of GUVs, a PcoB-containing and a protein-free vesicles were chosen, and micrographs were recorded in three channels. (A) Slice of vesicles, showing the fluorescent images in three channels: rhodamine-labeled lipids (red), Pacific Blue-labeled PcoB (blue), and FluoZin-3-Zn complex (green). (B) Z-stack of a PcoB-containing vesicle showing a nearly perfect spherical shape, with the same colors as in panel A. (C)Intensity profiles of the PcoB-containing and control GUVs along the dashed lines in (A), respectively. The colors correspond to the fluorescent dyes.

Figure 3

Flux curves for proteoliposomes subjected to copper delivered using a pipette. (A) Representative GUVs recorded in 5 s intervals for vesicles incorporated with wild-type or E252A PcoB or with empty GUVs as a control. The sizes of the vesicles were estimated to be 5–7 μm in diameter. (B) Time course of the Cu flux, 0.5 μL of 1 M CuCl₂ was supplemented to one side of the channel slide. PcoB-containing GUVs (black circles, n = 3 vesicles from one representative experiment), PcoB mutant E252A-containing GUVs (red circles, n = 7), and control vesicles (gray, n = 8) are shown. Error bars represent standard deviations. The vesicles were from the same batch.

Figure 4

Flux curves for proteoliposomes subjected to copper delivered using a syringe pump. (A) Representative GUVs recorded in 100 s intervals for vesicles incorporated with wild-type PcoB. The shown vesicles were estimated to be about 5 μm in diameter. (B) Time course of the Cu flux, 1 mM (black circles, n = 5) or 0.5 mM (gray circles, n = 6) were supplemented to the GUVs. A control sample with 1 mM Cu and no protein reconstituted into GUVs is also shown (red circles, n = 4). Error bars represent standard deviations. The vesicles were from the same batch.