Abstract
Abstract: :
Purpose:The goal of the present study is to assess the effect of prevalent C-terminal posttranslational modifications of aquaporin 0 (AQP0), observed in normal aging and cataractous human lenses, on the water permeability of AQP0 and potential regulation of AQP0 water channel activity. Methods:The water permeabilities of human wild type and mutant AQP0's, 1-234, 1-238, 1-243, S235A, were measured in a Xenopus laevis oocyte swelling assay. A direct comparison of wild type and mutant AQP permeability was permitted by the development of a new extracellular binding assay to quantitate the level of AQP0 protein membrane expression. The effects of kinase stimulation on the permeability were examined following treatment of oocytes with PMA or forskolin. Results:A dose dependent increase in water permeability and extracellular binding was observed in Xenopus oocytes injected with increasing amounts of wt, 1-243, and S235A AQP0 mRNA. Truncation of AQP0 at residue 243 resulted in a lower water permeability than that observed for the wild type protein (5.2 x10-3 and 6.6 x10-3 cm/sec). However, after normalizing for membrane expression, the permeability per molecule for the truncated protein, 1-243, was the same as for wild type AQP0. Further truncation of AQP0 at residues 234 and 238 prevented the incorporation of 1-234 and 1-238 into the oocyte plasma membrane. Activation of PKC by 100nM PMA resulted in a 10-25% decrease in the permeability of oocytes injected with wild type or S235A AQP0 mRNA. Conclusion:Truncation of the C-terminal residues 244-263 from AQP0, a major modification in aged human lenses, does not alter the water channel activity of AQP0. The lack of membrane incorporation of truncated AQPs, 1-234 and 1-238, precluded the measurement of channel activity and indicates that the C-terminus of AQP0 is necessary for protein trafficking. Phosphorylation may play a role in regulation of channel activity indirectly or through a site other than S235 as suggested by the decrease in the permeability of oocytes expressing wild type and S235A AQP0 after PKC stimulation.
Keywords: 527 protein structure/function • 525 protein modifications-post translational • 309 aging