TABLE 2.
Relative membrane expression of AQP4 phosphorylation mutants in response to hypotonic stimulus
Translocation of phosphomimetic and phospho-blocking mutants of AQP4-GFP in HEK293 cells, measured by confocal microscopy and subsequent image analysis, is shown. Cells were imaged before and after reduction of the extracellular osmolality to 85 mosm/kg of H2O. Each RME value is an average over three independent experiments, with each experiment analyzing at least 3 cells/micrograph and 3 line profiles/cell. Constitutive surface expression was measured by cell surface biotinylation and normalized to wild-type expression. p < 0.05 was considered statistically significant (*). Abbreviation: NA, not applicable.
| Mutant | Isotonic RME (± S.E.) | Hypotonic RME (± S.E.) | Mean % of area change (± S.E.) | Translocation | Constitutive surface expression (% of wild-type) |
|---|---|---|---|---|---|
| S52A | 34.18 (4.06) | 59.34 (5.53)* | 127.03 (5.99)* | Yes | 98.1 (5.3) |
| S52D | NA | NA | NA | No | 17.0 (6.3)* |
| S111A | 36.24 (9.95) | 62.43 (5.42)* | 130.69 (4.76)* | Yes | 94.8 (6.4) |
| S111D | 31.17 (4.22) | 52.99 (5.41)* | 120.62 (8.40)* | Yes | 91.4 (4.6) |
| S180A | 35.43 (7.78) | 69.03 (6.94)* | 142.65 (7.78)* | Yes | 109.8 (4.5) |
| S180D | 36.86 (6.07) | 53.56 (4.96)* | 148.16 (16.74)* | Yes | 99.7 (5.0) |
| S188A | 34.17 (7.21) | 63.31 (7.00)* | 137.01 (9.17)* | Yes | 98.6 (3.2) |
| S188D | 31.64 (9.11) | 59.18 (5.60)* | 135.53 (10.47)* | Yes | 104.1 (4.2) |
| S276A | 36.61 (4.64) | 36.97 (5.53) | 110.82 (3.85) | No | 96.8 (3.2) |
| S276D | 25.72 (5.42) | 57.93 (11.01)* | 134.18 (15.10)* | Yes | 83.5 (5.7)* |
| S276D + PKA inhibitor | 24.58 (4.85) | 51.14 (5.25)* | 127.12 (7.34)* | Yes | 86.2 (5.1)* |