Abstract
Purpose: :
To determine the role of calmodulin in the regulation of water permeability of AQP0. WT AQP0 is a low water permeability channel whose water permeability can be increased with low Ca2+ concentration and acidic pH in Xenopus oocyte expression system. Mutations in the calmodulin binding site resulted in two major phenotypes: low water permeability (low-Pf) or high water permeability channels (high-Pf). Two low water permeability mutants show a unique 5 mM Ca2+ regulation (S235D and S241A). We studied the molecular interactions between calmodulin and AQP0, and tested the effect of calmodulin on AQP0 water permeability under various conditions.
Methods: :
cRNAs were expressed in Xenopus oocytes and the water permeability was measured in response to an osmotic challenge in various calcium and pH conditions. AQP0 and its mutants were expressed in the presence of calmodulin inhibitors, PKA and PKC activators, or co-expressed crippled calmodulin. We measured the Fluorescent Resonance Energy Transfer (FRET) between fluorescently labeled calmodulin (CaM-488) and a fluorescently labeled antibody directed against the AQP0 C-terminus.
Results: :
In the presence of calmodulin inhibitors AQP0 had high Pf but co-expressing AQP0 with crippled calmodulin (that is unable to bind Ca2+) locked the water permeability in a low Pf state. Also, when S235D and R241A mutants were co-expressed with crippled calmodulin, the calcium regulation was lost and the channel was locked in the low Pf state. These two mutants were transformed into high-Pf channels upon additional phosphorylation (incubation with PKC activators), whereas the wild channel was locked into the low Pf state under the same conditions. These data suggest that calmodulin may dock on the C-terminus in two different positions. We further investigated the association of calmodulin with both wild type and mutant AQP0 using the FRET technique. We found that in the case of wild-type AQP0 the fluorescently labeled calmodulin (CaM-488) was able to form FRET with the fluorescently labeled C-terminal AQP0 antibody (Ab-AQP0E-555).
Conclusions: :
Calmodulin acts as an inhibitor of the water permeability of AQP0. The binding of calmodulin to the C-terminus results in a low-Pf channel that can be regulated with Ca2+ or pH. Calmodulin binding may happen in more then one configuration depending on the phosphorylation of AQP0 C-terminus.
Keywords: ion channels • cell membrane/membrane specializations • cataract