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J. Biol. Chem., Vol. 281, Issue 23, 15959-15969, June 9, 2006

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Identification of Key Functional Domains in the C Terminus of the K⁺-Cl^– Cotransporters^*

From the Nephrology Research Group, L'Hôtel-Dieu de Québec Institution, Department of Medicine, Faculty of Medicine, Laval University, Québec G1R 2J6, Canada

The K⁺-Cl^– cotransporter (KCC) isoforms constitute a functionally heterogeneous group of ion carriers. Emerging evidence suggests that the C terminus (Ct) of these proteins is important in conveying isoform-specific traits and that it may harbor interacting sites for 4-phorbol 12-myristate 13-acetate (PMA)-induced effectors. In this study, we have generated KCC2-KCC4 chimeras to identify key functional domains in the Ct of these carriers and single point mutations to determine whether canonical protein kinase C sites underlie KCC2-specific behaviors. Functional characterization of wild-type (wt) and mutant carriers in Xenopus laevis oocytes showed for the first time that the KCCs do not exhibit similar sensitivities to changes in osmolality and that this distinguishing feature as well as differences in transport activity under both hypotonic and isotonic conditions are in part determined by the residue composition of the distal Ct. At the same time, several mutations in this domain and in the proximal Ct of the KCCs were found to generate allosteric-like effects, suggesting that the regions analyzed are important in defining conformational ensembles and that isoform-specific structural configurations could thus account for variant functional traits as well. Characterization of the other mutants in this work showed that KCC2 is not inhibited by PMA through phosphorylation of its canonical protein kinase C sites. Intriguingly, however, the substitutions N728S and S940A were seen to alter the PMA effect paradoxically, suggesting again that allosteric changes in the Ct are important determinants of transport activity and, furthermore, that the structural configuration of this domain can convey specific functional traits by defining the accessibility of cotransporter sites to regulatory intermediates such as PMA-induced effectors.

Received for publication, January 3, 2006 , and in revised form, March 23, 2006.

^* This work was supported by the Kidney Foundation of Canada and the Canadian Institute of Health and Research (Grants MOP-68949 and MOP-15405). 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 Fonds de la Recherche en Santé du Québec scholars.

² A professor of Medicine at Laval University and holder of a Canada Research Chair in Molecular Physiology. To whom correspondence should be addressed: L'Hôtel-Dieu de Québec du CHUQ, 10 Rue McMahon, Québec G1R2J6, Canada. Tel.: 418-691-5151 (ext. 15477); Fax: 418-692-5795; E-mail: paul.isenring{at}crhdq.ulaval.ca.

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