Movement of water through very narrow membrane channels is different from Poiseuillian flow through macroscopic tubes. The diffusive nature of single-file Transport and the unaltered fluidity of confined water suggest that the unitary osmotic water channel permeability pf may only vary within the narrow limits that are set by the small length deviations of the single-file region. This is in sharp contrast to the experimental observation that pf alterations stretch over several orders of magnitude. To rationalize this variability, we now introduced major improvements to the technical design of pf measurements: (i) We counted the number of reconstituted channels per unit of membrane area by using single molecule techniques, i.e. we attached a dye to each
channel and monitored its fluorescence and we used an atomic force microscope, which like a gramophone, scratched over the surface of the membrane and thereby detected small height differences between the protein and the surrounding lipid; (ii) We used a
theory-based approach that links the water efflux from small vesicles to the intensity of scattered light; and (iii) We prevented osmolyte dilution within the near-membrane
stagnant water layers from interfering with our measurements. These improvements enabled us (i) to show that previously pf of aquaporins was 5 to 15-fold underestimated and (ii) to find the determinants of water mobility in narrow membrane channels.
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