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J. Biol. Chem., Vol. 282, Issue 46, 33475-33483, November 16, 2007

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Epithelial Sodium Channel Exit from the Endoplasmic Reticulum Is Regulated by a Signal within the Carboxyl Cytoplasmic Domain of the Subunit^*

Gunhild M. Mueller, Ossama B. Kashlan, James B. Bruns, Ahmad B. Maarouf, Meir Aridor, Thomas R. Kleyman¹, and Rebecca P. Hughey

From the Renal-Electrolyte Division, Department of Medicine, and Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Epithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from , , and subunits, each with two transmembrane domains, a large extracellular loop, and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the and subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with -glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared with control Tac or GGT, but we did observe enhanced exit of Tac-Ct when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved as cleaved subunit, but not non-cleaved , is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated (624–699-V5) with full-length was similar to wild type (+), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of (613–699-V5 +) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence ⁶¹³HRFRSRYWSPG⁶²³ within the context of the full-length revealed that mutation RSRYW⁶²⁰ to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the subunit carboxyl cytoplasmic tail.

Received for publication, August 31, 2007 , and in revised form, September 11, 2007.

^* This work was supported by National Institutes of Health Grant DK65161 (to T. R. K. and R. P. H.). 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 To whom correspondence should be addressed: Renal-Electrolyte Division, Dept. of Medicine, University of Pittsburgh School of Medicine, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-647-3121; Fax: 412-648-9166; E-mail: kleyman{at}pitt.edu.

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