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J. Biol. Chem., Vol. 283, Issue 37, 25290-25295, September 12, 2008

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Proteolytic Processing of the Epithelial Sodium Channel Subunit Has a Dominant Role in Channel Activation^*

Marcelo D. Carattino, Rebecca P. Hughey¹, and Thomas R. Kleyman

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

Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the subunit and at a single extracellular site within the subunit. Channels lacking furin processing of the subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK¹⁸⁶) in the subunit. We also demonstrated that the tract D206-R231, between the two furin cleavage sites in the subunit, as well as the tract E144-K186, between the furin and prostasin cleavage sites in the subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the and subunit inhibitory tracts, respectively. We examined whether release of either the or inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the subunit (R205A,R208A,R231Aβ) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of the subunit, amiloride-sensitive currents in oocytes expressing channels carrying subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (βR143A,E144-K186 and R205A,R208A,R231AβR143A, E144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the and subunit inhibitory tracts, subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in the subunit (βR143A,E144-K186) showed a significant reduction in the efficacy of block by the synthetic-26 inhibitory peptide representing the tract D206-R231. Our data indicate that removal of the inhibitory tract from the subunit, in the absence of subunit cleavage, results in nearly full activation of the channel.

Received for publication, May 22, 2008 , and in revised form, July 15, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01/R56 DK065161 and P30 DK079307. This work was also supported by the Cystic Fibrosis Foundation (R883CR02 and Kleyma08P0). 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.

We dedicate this report to the memory of James B. Bruns.

¹ To whom correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, S933 Scaife Hall, 3550 Terrace St, Pittsburgh, PA 15261. Tel.: 412-383-8949; Fax: 412-383-8956; E-mail: hugheyr{at}pitt.edu.

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