Quantification of Water Flux in Large Unilamellar Vesicles

Abstract

Water homeostasis is fundamental to all forms of life and plays a major role in human health and disease, plant growth or bacterial survival. It is balanced through a complex interplay of osmotically active substance across lipid membranes and narrow membrane channels. The endeavors to understand the determinants of water permeation and the effect of the lipid or polymer membrane on channel function, the development of specific water flow inhibitors, the design of artificial water channels and aquaporins for the use in industrial water filtration applications rely on accurate ways to quantify water permeabilities (Pf). A commonly used method is to reconstitute membrane channels into large unilamellar vesicles (LUVs) and to subject these vesicles to an osmotic gradient in a stopped-flow device. Fast recordings of either scattered light intensity or fluorescence self-quenching signals are taken as a readout for vesicle volume change, which in turn can be recalculated to accurate Pf values (Horner at al., Science Advances 1, e1400083, 2015; Hannesschläger et al., Sci Rep 8, 8516, 2018). By means of computational and experimental data, we discuss the pros and cons of using scattering versus self-quenching experiments or subjecting vesicles to hypo- or hyperosmotic conditions. In addition, we explicate the influence of the LUVs size distribution and remaining detergent after protein reconstitution on Pf values. We point out that results such as that the single channel water permeability (pf) depends on the membrane matrix or on the direction of the applied osmotic gradient may be direct results of the measurement and analysis procedure.