Effect of Oligomerization and Phosphorylation on the Unitary Osmotic Water Channel Permeability
Sprache des Vortragstitels:
Second World Congress on Water Channel Proteins (Aquaporins and Relatives)
Sprache des Tagungstitel:
Introduction. Channel function may depend on posttranslational modifications and oligomerization: Aquaporins form tetramers although the monomer acts as the water conducting unit. Some of the water channels undergo phosphorylation. The physiological function of the tetrameric assembly is unknown and the functional consequences of phosphorylation are debated. Here we investigate possible effects that (i) cooperativity in AQP1 and GlpF or (ii) phosphorylation of two AQP4 residues may have on the unitary water channel permeability pf.
Materials and Methods. AQPs are purified and reconstituted into artificial lipid vesicles. Subsequently, we osmotically deflate the vesicles. Using our new adaptation of the Rayleigh-Gans-Debye equation (1), we calculate the water efflux from the accompanying increase in the intensity of scattered light. Fluorescence correlation spectroscopy serves to determine the number of fluorescently labeled channels per vesicle. Therefore we count the number of particles before and after dissolving the proteoliposomes by a mild detergent. High resolution AFM images confirm the concentration dependent presence of monomers, dimers and trimers in our AQP1 and GlpF proteoliposome samples. Quantification of both water efflux and channel number per vesicle allow calculation of pf.
Results and Discussion. AQP1 and GlpF reconstitute as functional monomers, dimers, trimers and tetramers. pf does not dependent on their oligomeric state. Likewise, point mutations that mimic AQP4 phosphorylation at position S111 and S180 have no effect on pf.
Conclusions. Neither tetrameric assembly of AQP1 and GlpF nor phosphorylation of AQP4 alter the mobility of the single-file water molecules.
Keywords. cooperativity, phosphorylation, aquaporins, water, permeability
Acknowledgements. This work was supported by grant P23466 of the Austrian Science Fund.
References. 1. Horner et al. Sci. Adv. 2015;1:e1400083
Topic: Water Channel Proteins in Reconstituted Systems