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J. Biol. Chem., Vol. 283, Issue 30, 21278-21283, July 25, 2008

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Phosphorylation Determines the Calmodulin-mediated Ca²⁺ Response and Water Permeability of AQP0^*

From the Department of Physiology and Biophysics, University of California, Irvine, California 92697-4561

In Xenopus oocytes, the water permeability of AQP0 (P_f) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca²⁺ regulation of P_f, we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P_f manifests four distinct phenotypes: Group 1, with high P_f upon removal of external Ca²⁺ (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P_f in elevated (5 mM) external Ca²⁺ (S235D and R241A); Group 3, with high P_f and no Ca²⁺ regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P_f and no Ca²⁺ regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P_f and no Ca²⁺ regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P_f. CaM binding is associated with a low P_f state, and a lack of CaM binding is associated with a high P_f state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P_f changes contributing to the development of a cataract.

Received for publication, March 4, 2008 , and in revised form, May 16, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant EY 5661 (to J. E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this manuscript.

² To whom correspondence may be addressed. Tel.: 949-824-6676; E-mail: kkalman{at}uci.edu.

³ To whom correspondence may be addressed. Tel.: 949-824-5835; Fax: 949-824-3143; E-mail: jhall{at}uci.edu.

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