Effect of oligomerization on the unitary water permeability of AQP4 reconstituted into giant unilamellar vesicles

Description

Danila Boytsov, Andreas Horner, Christof Hannesschlaeger, Christine Siligan and Peter Pohl Johannes Kepler University Linz, Institute of Biophysics The formation of larger membrane protein oligomers may have a profound effect on protein function. For example, the formation of arrays of aquaporin-4 molecules was hypothesized to be responsible for the high water permeability observed in overexpressing cells. It was thus far impossible to test this hypothesis in a reconstituted system, because only a small number of AQP4 molecules could be reconstituted into large unilamellar vesicles (LUVs). Here we show that protein reconstitution into giant unilamellar vesicles (GUVs) provides a solution. Instead of assessing the volume change from the intensity of scattered light as performed with LUVs1, we implement the micropipette aspiration technique. It offers the possibility to measure small changes in relative volume. Additional advantages are that membrane channel abundancy and the oligomeric state are directly determined by fluorescence correlation spectroscopy in the very same GUV from which the volume flow is extracted. We tested the new assay by reconstituting the M1 and M23 isoforms of AQP4 which are known to remain in their homotetrameric state or to form orthogonal arrays, respectively. We found it mandatory to properly account for unstirred layer effects, i.e. for osmolyte and solute dilution adjacent to the external surface of the GUV and for solute concentration increases in the immediate vicinity of the internal leaflet. We also investigated the effect of S111 and S180 phosphorylation on array formation and the unitary AQP4 water permeability. 1. Horner et al. (2015). Science Advances 1, e1400083.

Period	22 Sept 2016
Event title	8th ÖGMBT Annual Meeting 2016 - Life Sciences for the Next Generation
Event type	Conference
Location	AustriaShow on map