Table 1.
Crystallographic data and refinement statistics for full-length and truncated hAQP5 S156E structures.
Fig 1.
Surface expression of AQP5 S156 mutants. Representative fluorescence confocal micrographs of HEK293 cells transfected with GFP-tagged (A) AQP5 wild-type, (B) AQP5-S156A and (C) AQP5-S156E; the fluorescence intensity profiles along each yellow line are shown. (D) Relative membrane expression calculated from fluorescence intensity profiles: 5 profiles were taken per cell and at least 3 cells per micrograph, repeated in 3 independent experiments. Asterisks denote p < 0.05 using Student’s t-test followed by Bonferroni correction for multiple comparisons.
Fig 2.
Effect of PKA inhibition on surface expression of AQP5.
Representative fluorescence confocal micrographs of HEK293 cells transfected with GFP-tagged (A) AQP5 wild-type, (B) AQP5-S156A and (C) AQP5-S156E that were treated with a PKA inhibitory peptide for 30 minutes. (D) Relative membrane expression of the 3 AQP5 constructs with and without PKA inhibition. Asterisks denote p < 0.05 using Student’s t-test followed by Bonferroni correction for multiple comparisons.
Fig 3.
Hypotonicity-induced translocation of AQP5.
Relative membrane expression of AQP5 and mutants was measured in the same cells before and 1 minute after reduction of the extracellular osmolality to 85 mOsm/kg H2O by fourfold dilution of the culture medium with dH2O. PKA inhibition was achieved by a 30 minute incubation with a PKA inhibitory peptide. Asterisks denote p < 0.05 by paired t-tests followed by Bonferroni correction for multiple comparisons.
Fig 4.
Overall structure of AQP5 S156E. (A) The structure of full length (green, PDB code 4DYE) and truncated S156E AQP5 (orange, PDB code 5CX6) overlaid on the wild-type structure of AQP5 in blue (PDB code 3D9S). Water molecules in the water-conducting channel of truncated S156E AQP5 are shown as red spheres. (B) Same as in (A), viewed from the cytoplasmic side. (C) Structure of the carboxy-terminus of full-length S156E monomer D, showing its interactions with a symmetry-related molecule (grey). 2Fobs-Fcalc electron density is displayed at 1.0 σ. Loop D and Glu 156 in monomers A and D are highlighted in yellow. (D) Lipid molecule in the tetrameric channel of full-length and truncated AQP5 S156E.
Fig 5.
Structure of loop D and its interaction with the carboxy-terminus.
Wild-type AQP5 and AQP5 S156E are coloured blue and orange respectively. (A) Zoomed in view of the boxed area in Fig 4A showing that the interactions between loop D and the carboxy-terminus are maintained in the S156E mutant structure. Hydrogen bonds are shown as dashed lines. Structural comparison of (B) loop D and (C) the carboxy-terminus shows that there are no structural differences between wild-type AQP5 and AQP5 S156E. In (D), the four monomers from the crystal structure of human AQP2 are overlaid, showing a significant conformational variability of the carboxy-terminal helix within the tetramer. The four AQP2 monomers are colored in different shades of grey. (D) Structure of the S156E mutation site showing 2Fobs-Fcalc electron density contoured at 1.0 σ. The structure of wild-type AQP5 is shown in blue for comparison.
Fig 6.
A proposed model of the equilibrium between vesicular and surface-localized AQP5.
Panel A shows wild-type AQP5 under isotonic conditions. The large arrows represent an increase in AQP5 translocation (and the small arrows are a decrease). This is regulated by three independent factors: Phosphorylation of AQP5 at position S156 (orange cylinders in panel B; shown by a phosphomimetic glutamate substitution (S156E) of AQP5; the effect of PKA (green cylinders in panel C) and the effect of decreasing the relative tonicity of the environment (panel D). We speculate that these three pathways control the surface abundance of AQP5.