The measurement of ATPase activity has been the only assay available to study the effects of mutations on ABCA4 function. We employed this assay to examine the effects of [L541P; A1038V], R602W and C1490Y mutations on in vitro ATP hydrolysis. Constructs containing wild-type and mutated ABCA4 cDNAs, tagged with the eight amino acid bovine opsin C-terminal epitope (1D4), were expressed in COS7 cells and proteins were purified on a 1D4 affinity column. CHAPS-solubilized ABCA4 was incubated subsequently with ATP, and the hydrolysis rate was estimated with the charcoal method (31).The rate of ATP hydrolysis of the complex allele [L541P; A1038V] was decreased to 68.1% of wild-type ABCA4 (Fig. 3).

both the P and PV ABCA4 variants exhibited a dramatically reduced basal ATPase activity (∼30% of WT ABCA4), which did not increase upon the addition of all-trans-retinal. Thus, ABCA4 variants carrying the P mutation were functionally impaired.

Amounts of A2E in the eyes of Abca4PV/PV, Abca4−/− and WT mice at the ages of 1, 3, 6, 12 and 15 months were quantified by reverse-phase high-performance liquid chromatography (HPLC) (Fig. 9B). Mice were raised under a regular 12-h light (∼10 lux)/12-h dark cycle. Age-dependent A2E accumulation was noted in all genotypes, with Abca4PV/PV and Abca4−/− mice accumulating about 5-fold more A2E than WT mice. No statistically significant differences in A2E accumulation were found between Abca4PV/PV and Abca4−/− animals.