The two main pathways for plasma‐membrane water transport are either through the lipid bilayer or water‐selective pores (aquaporins). Aquaporins are an interesting large family of water pores; some isoforms are water‐selective, whereas others are permeable to small solutes. Aquaporin-1, the best-studied isoform, is present in the membrane as a tetramer. The precise quantification of facilitated water transport through membranes is notoriously difficult. In contrast to the ease with which the stepwise increment in current that is induced by the opening of a single ion channel can be detected, the contribution of a single water channel to the total water flux across the membrane usually goes unnoticed. In essence, Previous research in the field showed that a single channel water permeability, pf, can generally be gathered by measuring the water flux through an ensemble of channels. Nonetheless, the associated technical difficulties give rise to a large scatter of published pf values that covers more than one order of magnitude— even for a single aquaporin. This scatter has been associated with the presence of unstirred layers adjacent to all membranes. Attempts to resolve this and other pitfalls have proven difficult, but this has not deterred researchers. In a recent attempt, researchers employed the micropipette aspiration technique (MAT), which did little to improve the situation, since even the water permeability of the lipid matrix itself was reported to largely depend on the assumed unstirred layer thickness. Nonetheless, this technique has superb sensitivity that could allow one to resolve pf values with better accuracy.
In general, measurements of the unitary hydraulic conductivity of membrane channels, pf, may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for pf measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. To address this, Johannes Kepler University researchers: Danila Boytsov, Dr. Christof Hannesschlaeger, Professor Andreas Horner, Dr. Christine Siligan and Professor Peter Pohl, proposed to adopt the MAT technique credit to its superb sensitivity, and combine it with a fast perfusion system and fluorescence correlation spectroscopy, with the goal of developing an assay to measure pf of membrane channels reliably. Their work is currently published in the research journal, Biotechnology Journal.
In their approach, protein density was derived from intensity fluctuations that fluorescently labeled channels in the aspirated giant unilamellar vesicles induced by diffusion through the diffraction limited spot. The researchers started by engaging in overexpression, purification and reconstitution of the aquaporin-1. Next, giant unilamellar vesicles containing aquaporin-1 were prepared using a modified electro formation protocol.
The authors reported that by combining MAT with a fast perfusion system and fluorescence correlation spectroscopy, they were able to take measurements of protein abundance, unstirred layers limitations, and the decrement in the giant unilamellar vesicles volume in a single experiment. To be specific, the perfusion system was seen to minimize unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. Remarkably, pf amounted to 2.4 ± 0.1 × 10−13 cm³ s−1 for aquaporin-1 that served as a test case.
In summary, the study successfully demonstrated the adoption of the micropipette aspiration technique for measurements of pf. Generally, the team reported that micro-aspiration was well suited for use in determining the pf values of membrane channels, since its superb sensitivity offers the possibility to investigate proteins with inherent low reconstitution efficiency. In a statement to Medicine Innovates, Professor Peter Pohl explained their new developed assay paves the way for one to directly monitor the effect that interaction of aquaporins with other proteins or inhibitors may have on pf, on a single sample.
Danila Boytsov, Christof Hannesschlaeger, Andreas Horner, Christine Siligan, Peter Pohl. Micropipette Aspiration-Based Assessment of Single Channel Water Permeability. Biotechnology Journal 2020, volume 15, 1900450.Go To Biotechnology Journal