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J. Biol. Chem., Vol. 283, Issue 13, 8711-8722, March 28, 2008

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Congenital Chloride-losing Diarrhea Causing Mutations in the STAS Domain Result in Misfolding and Mistrafficking of SLC26A3^*

Michael R. Dorwart, Nikolay Shcheynikov, Jennifer M. R. Baker^¶||, Julie D. Forman-Kay^¶||, Shmuel Muallem, and Philip J. Thomas¹

From the Department of Physiology and the Molecular Biophysics Program, University of Texas Southwestern Medical Center, Dallas, Texas 75390, ^¶Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario M5G1X8, Canada, and the ^||Department of Biochemistry, University of Toronto, Toronto, Ontario M5S1A8, Canada

Congenital chloride-losing diarrhea (CLD) is a genetic disorder causing watery stool and dehydration. Mutations in SLC26A3 (solute carrier 26 family member 3), which functions as a coupled Cl^-/ exchanger, cause CLD. SLC26A3 is a membrane protein predicted to contain 12 transmembrane-spanning -helices and a C-terminal STAS (sulfate transporters and anti-sigma-factor) domain homologous to the bacterial anti-sigma-factor antagonists. The STAS domain is required for SLC26A3 Cl^-/ exchange function and for the activation of cystic fibrosis transmembrane conductance regulator by SLC26A3. Here we investigate the molecular mechanism(s) by which four CLD-causing mutations (Y526/7, I544N, I675/6ins, and G702Tins) in the STAS domain lead to disease. In a heterologous mammalian expression system biochemical, immunohistochemical, and ion transport experiments suggest that the four CLD mutations cause SLC26A3 transporter misfolding and/or mistrafficking. Expression studies with the isolated STAS domain suggest that the I675/6ins and G702Tins mutations disrupt the STAS domain directly, whereas limited proteolysis experiments suggest that the Y526/7 and I544N mutations affect a later step in the folding and/or trafficking pathway. The data suggest that these CLD-causing mutations cause disease by at least two distinct molecular mechanisms, both ultimately leading to loss of functional protein at the plasma membrane.

Received for publication, May 25, 2007 , and in revised form, December 21, 2007.

^* This work was supported by National Institutes of Health Grants NIDDK 49835 (to P. J. T.), DE12309, and DK38938 (to S. M.), Canadian Institutes of Health Research Grant 145629 (to J. D. F.-K.), and National Institutes of Health Training Grant GM-08203 (to M. R. D.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed. E-mail: Philip.Thomas{at}utsouthwestern.edu.

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