Fig 2: Both AQP4 isoforms are relocalized. Both the M1 and the M23 isoforms of AQP4 are translocated to the cell surface upon hypotonic stimulus. A, membrane organization of AQP4-GFP transfected into HEK293 cells. The M1 isoform has a homogeneous membrane distribution, whereas M23 clusters into orthogonal arrays of particles. B, SDS-PAGE of protein transcribed in HEK293 cells from the AQP4 wild-type mRNA, AQP4 M1 construct, and AQP4 M23 construct. No M23 protein was detected in the wild-type sample. C, there was no significant difference in constitutive surface expression between the three constructs. D, both isoforms of AQP4 are relocalized in response to reduction of the extracellular osmolality to 85 mosm/kg of H2O (M1: p = 0.002, n = 3. M23: p = 0.004, n = 3. WT: p = 0.001, n = 3.). All data are presented as mean ± S.E.

Fig 3: Cell swelling of AQP4-transfected cells. AQP4-GFP-transfected HEK293 cells were imaged using the GFP tag and FM4-64, a fluorescent membrane marker. A, two-color fluorescence micrograph of AQP4-GFP-transfected cells (green) loaded with FM4-64 (red), before and after reduction of extracellular osmolality from 340 mosm/kg of H2O to 85 mosm/kg of H2O. B, representative cross-sectional areas of transfected cells before and after hypotonic challenge, calculated using a particle detection algorithm. The post-hypotonic challenge area as a percentage of the pre-challenge area is shown between each pair of images. C, representative cross-sectional areas of non-transfected cells from the same image.

Fig 4: Water permeability of non-tetrameric mutants. A, representative calcein fluorescence quenching curves from stably transfected MDCK cells subjected to a 200 mosmol of mannitol osmotic gradient. B, water permeability of MDCK cells normalized to AQP4 WT-transfected MDCK cells. C, normalized surface expression of AQP4 constructs in the stably expressing MDCK clones used for water permeability measurements, measured by cell surface biotinylation. D, MDCK membrane water permeability normalized to surface expression, to give normalized single channel permeability. All data are presented as mean ± S.E., n = 4.

Fig 5: Tonicity-induced translocation of non-tetrameric mutants. A, representative fluorescence micrographs of HEK293 cells transfected with AQP4-GFP fusion proteins, before and after 30 s of exposure to hypotonic (85 mosmol) medium. B, relative membrane expression of AQP4-GFP fusion proteins before and after exposure to hypotonic medium. At least 4 cells per image were analyzed for each experimental repeat, n = 3. Data are presented as mean ± S.E.