Abstract
Purpose: :
To examine the calmodulin regulation of the water permeability of wild type and mutant AQP0s in Xenopus oocytes and the interaction between calmodulin and the cytoplasmic surface of AQP0.
Methods: :
Site directed mutagenesis, BtAQP0, Xenopus oocyte water permeability assay, calmodulin, Western blot, confocal microscopy.
Results: :
In Xenopus oocytes, the water permeability of AQP0 (Pf) increases in o mM external calcium, an effect mediated by cytoplasmic calmodulin (CaM) bound to the C-terminus of AQP0. In the current model of water permeability regulation of AQP0, CaM binding to C-terminus of AQP0 is associated with a low Pf state, and a lack of CaM binding is associated with a high Pf state. AQP0 enters into a "locked-low" phenotype when PKA, PKC enzymes are up-regulated and when Serines at 229 and 235 positions are mutated to Asparagine to mimic phosphorylation. We hypothesize that in the locked-low state of AQP0 calmodulin interacts differently with the cytoplasmic surface of AQP0. We demonstrate that CaM can have at least two binding configuration with AQP0 depending on the phosphorylation state of S235 and the presence of apo-CaM and Ca2+-bound CaM. In Xenopus oocytes, when crippled CaM (which cannot bind Ca2+) binds to AQP0, the water permeability switches into a low-Pf phenotype similar to that induced by wild-type CaM-AQP0 binding. In the presence of crippled-CaM,0 mM Ca2+ does not Pf but 5 mM Ca2+ does. The acid pH induced Pf increase remained intact even when crippled CaM was present. When S235D-AQP0 was co-expressed with crippled CaM, both 0 mM and 5 mM Ca2+ regulation of Pf were eliminated but acid pH induced Pf increase was unaffected.
Conclusions: :
The binding configuration of calmodulin determines AQP0 water permeability and mode of regulation. Crippled-CaM binds to both wild-type and S235D-AQP0 resulting in two distinct phenotypes. The variability in phenotypes could reflect additional, distinct interactions with the cytoplasmic surface of AQP0.
Keywords: calcium • ion channels • cataract