TABLE 1:
Parameters of ATP8A2 C-terminal domain mutants determined in this study.
| ATPase activity anti-1D4 column | ATPase activity anti-7F4 column | Neurite length in PC12 cells | [Ca2+] inhibition | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ATP8A2 | n | Vmax (±SEM) μmol Pi/min per mg | KA (±SEM) μM | n | Vmax (±SEM) μmol Pi/min per mg | KA (±SEM) μM | n | Increase in neurite length (±SEM) µm | n | IC50 (±SEM) mM |
| WT | 18 | 136.8 (10.24) | 25.4 (3.11) | 5 | 126.7 (15.08) | 38.5 (5.01) | 121 | 51.21 (3.9) | 6 | 2.72 (0.67) |
| −20 | 6 | 95.03 (16.08) | 22.54 (2.8) | 3 | 55.5 (4.8) | 37.2 (4.6) | 58 | 32.9 (4.2) | 3 | 1.01 (0.02) |
| −33 | 6 | 81.66 (17.32) | 19.51 (2.65) | 3 | 50.36 (2.3) | 29.8 (6.6) | 63 | 4.9 (2.06) | 2 | 0.93 (0.4) |
| −60 | 6 | 108.14 (11.8) | 18.14 (3.15) | 3 | 59.5 (15.8) | 21.6 (0.93) | 105 | 18.4 (4.0) | 2 | 1.37 (0.32) |
| −80 | 4 | 155.93 (12.82) | 20.68 (1.57) | 3 | 85.06 (13.3) | 32.4 (5.9) | 112 | 37.01 (3.53) | 2 | 1.99 (0.4) |
ATPase activity of the different ATP8A2 mutants was titrated against increasing concentration of PS in the presence of 0.5 mM ATP to determine the maximal reaction rate (Vmax) and half-maximum activation constant (KA). Increase in the length of neurites formed by differentiated PC12 overexpressing the different ATP8A2 mutants compared with untransfected cells was measured. IC50 for inhibition of ATPase activity of different ATP8A2 mutants was estimated by titrating activity in the presence of increasing CaCl2 and fixed PS and ATP concentrations. n is the number of independent measurements.