Effects of Calmodulin/AQP0 Interaction on the Calcium Sensitivity of AQP0 Water Permeability

Abstract

Purpose:: To investigate the role of calmodulin (CaM) and the phosphorylation of the C-terminus on the calcium sensitivity of AQP0 water permeability (P_f).

Methods:: We expressed AQP0 mutants, which mimicked phosphorylation at serines of the CaM-binding site, along with wild-type AQPO in Xenopus oocytes. We measured P_f in response to an osmotic challenge at different calcium concentrations (µM, 2 or 5 mM) in the presence of CaM inhibitors (TFP, CDZ and W7) or crippled CaM (which is unable to bind calcium). Endogenous Xenopus CaM was present in all experiments. We used confocal microscopy to image the co-localization of a fluorescent CaM(Cam488) in the oocyte membrane with either AQP0 or its mutants.

Results:: The low and high P_f state of AQP0 depends on pH, Ca²⁺ concentration, and the presence of calmodulin. In the presence of CaM inhibitors AQP0 lost its Ca²⁺ regulation and was locked in a high P_f state. When crippled CaM and AQP0 were co-expressed, 5 mM Ca²⁺ rather than µM Ca²⁺ induced a high P_f. Interestingly, when Ser235Asp mutant was co-expressed with crippled CaM, its P_f remained low and lost its Ca²⁺ regulation. Other CaM-binding-site mutants (Ser229Asp, Ser231Asp, Ser231Asn and Ser229Asn/Ser235Asp) resulted in a high P_f channel. In the oocyte membrane we observed less fluorescence in the CaM488/Ser229Asp co-expression than in the CaM488/AQP0 co-expression.

Conclusions:: CaM inhibitors prevent any binding of CaM to AQP0, and crippled CaM binds to AQP0 but cannot sense the Ca²⁺ . The opposite effects of CaM inhibitors and crippled CaM on the P_f of AQP0 suggest that when CaM binds to AQP0 the P_f is low, and when CaM is released upon Ca²⁺ modulation the P_f is high. Phosphorylations of Ser229 and Ser231 result in a high P_f channel, possibly because of decreased calmodulin binding.

Keywords: cell membrane/membrane specializations • ion channels • cataract