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J. Biol. Chem., Vol. 279, Issue 7, 5648-5654, February 13, 2004
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From the Nephrology Research Group, Department of Medicine, Faculty of Medicine, Laval University, Québec G1R 2J6, Canada
The 2nd transmembrane domain (tm) of the secretory Na+-K+-Cl- cotransporter (NKCC1) and of the kidney-specific isoform (NKCC2) has been shown to play an important role in cation transport. For NKCC2, by way of illustration, alternative splicing of exon 4, a 96-bp sequence from which tm2 is derived, leads to the formation of the NKCC2A and F variants that both exhibit unique affinities for cations. Of interest, the NKCC2 variants also exhibit substantial differences in Cl- affinity as well as in the residue composition of the first intracellular connecting segment (cs1a), which immediately follows tm2 and which too is derived from exon 4. In this study, we have prepared chimeras of the shark NKCC2A and F (saA and saF) to determine whether cs1a could play a role in Cl- transport; here, tm2 or cs1a in saF was replaced by the corresponding domain from saA (generating saA/F or saF/A, respectively). Functional analyses of these chimeras have shown that cs1a-specific residues account for most of the A-F difference in Cl- affinity. For example, Km(Cl-)s were
8 mM for saF/A and saA, and
70 mM for saA/F and saF. Intriguingly, variant residues in cs1a also affected cation transport; here, Km(Na+)s for the chimeras and for saA were all
20 mM, and Km(Rb+) all
2 mM. Regarding tm2, our studies have confirmed its importance in cation transport and have also identified novel properties for this domain. Taken together, our results demonstrate for the first time that an intracellular loop in NKCC contributes to the transport process perhaps by forming a flexible structure that positions itself between membrane spanning domains.
Received for publication, October 13, 2003 , and in revised form, November 12, 2003.
* This work was supported by grants from the Canadian Institute of Health and Research (MT-15405) and the Kidney Foundation of Canada. 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.
A Fonds de la Recherche en Santé du Québec (FRSQ) scholar.
To whom correspondence should be addressed: L'Hôtel-Dieu de Québec Research Center, 10 Rue McMahon (Rm. 3852), Québec G1R 2J6, Canada. Tel.: 418-691-5151 (ext. 15477); Fax: 418-691-5787; E-mail: paul.isenring{at}crhdq.ulaval.ca.
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