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J. Biol. Chem., Vol. 281, Issue 25, 17266-17275, June 23, 2006

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Affinity-defining Domains in the Na-Cl Cotransporter

A DIFFERENT LOCATION FOR Cl^- AND THIAZIDE BINDING^*

Erika Moreno¹, Pedro San Cristóbal¹, Manuel Rivera, Norma Vázquez, Norma A. Bobadilla, and Gerardo Gamba²

From the Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México Tlalpan 14000, Mexico City, Mexico and Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo 42160, México

The thiazide-sensitive Na⁺-Cl^- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na⁺, Cl^-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different.

Received for publication, March 20, 2006 , and in revised form, April 10, 2006.

^* This work was supported by National Institutes of Health Grant DK-64635 and Wellcome Trust Grant GR070159MA (to G. G.). This study was presented in part at the 2005 Experimental Biology meeting in San Diego, CA, and at the 2005 Renal Week of the American Society of Nephrology, Philadelphia, PA. 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 These authors contributed equally to this work.

² To whom correspondence should be addressed: Molecular Physiology Unit, Vasco de Quiroga No. 15, Tlalpan 14000, Mexico City, Mexico. Tel.: 5255-5513-3868; Fax: 5255-5655-0382; E-mail: gamba{at}biomedicas.unam.mx or gamba{at}quetzal.innsz.mx.

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