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J. Biol. Chem., Vol. 281, Issue 2, 1016-1026, January 13, 2006

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A C-terminal Domain in KCC2 Confers Constitutive K⁺-Cl^- Cotransport^*

Adriana Mercado1, Vadjista Broumand, Kambiz Zandi-Nejad2, Alissa H. Enck, and David B. Mount3

From the Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 and the Division of General Internal Medicine, Veterans Affairs Boston Healthcare System, Harvard Medical School, West Roxbury, Massachusetts 02132

The neuron-specific K⁺-Cl^- cotransporter KCC2 plays a crucial role in determining intracellular chloride activity and thus the neuronal response to -aminobutyric acid and glycine. Of the four KCCs, KCC2 is unique in mediating constitutive K⁺-Cl^- cotransport under isotonic conditions; the other three KCCs are exclusively swelling-activated, with no isotonic activity. We have utilized a series of chimeric cDNAs to localize the determinant of isotonic transport in KCC2. Two generations of chimeric KCC4-KCC2 cDNAs initially localized this characteristic to within a KCC2-specific expansion of the cytoplasmic C terminus, between residues 929 and 1043. This region of KCC2 is rich in prolines, serines, and charged residues and encompasses two predicted PEST sequences. Substitution of this region in KCC2 with the equivalent sequence of KCC4 resulted in a chimeric KCC that was devoid of isotonic activity, with intact swelling-activated transport. A third generation of chimeras demonstrated that a domain just distal to the PEST sequences confers isotonic transport on KCC4. Mutagenesis of this region revealed that residues 1021-1035 of KCC2 are sufficient for isotonic transport. Swelling-activated K⁺-Cl^- cotransport is abrogated by calyculin A, whereas isotonic transport mediated by KCC chimeras and KCC2 is completely resistant to this serine-threonine phosphatase inhibitor. In summary, a 15-residue C-terminal domain in KCC2 is both necessary and sufficient for constitutive K⁺-Cl^- cotransport under isotonic conditions. Furthermore, unlike swelling-activated transport, constitutive K⁺-Cl^- cotransport mediated by KCC2 is completely independent of serine-threonine phosphatase activity, suggesting that these two modes of transport are activated by distinct mechanisms.

Received for publication, September 12, 2005 , and in revised form, November 4, 2005.

^* This work was supported in part by National Institutes of Health RO1 DK57708 (to D. B. M.). 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 Supported in part by grants from the Harvard-Mexico Foundation.

² Recipient of an American Heart Association Postdoctoral Fellowship Award 0225706T.

³ Supported by an Advanced Research Career Development Award from Veterans Affairs. To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, Rm. 542, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-525-5876; Fax: 617-525-5830; E-mail: dmount{at}rics.bwh.harvard.edu.

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