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J. Biol. Chem., Vol. 280, Issue 28, 26206-26215, July 15, 2005

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On the Interaction between Amiloride and Its Putative -Subunit Epithelial Na⁺ Channel Binding Site^*

Ossama B. Kashlan, Shaohu Sheng, 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

The epithelial Na⁺ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are Ser-583, Gly-525, and Gly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the and glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the subunit resulted in moderate (<5-fold) weakening of amiloride K_i. We investigated the role of Ser-583 in amiloride binding by systematically mutating Ser-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a -methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at 583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some Ser-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that Ser-583 may have a role in ion permeation through ENaC.

Received for publication, March 30, 2005 , and in revised form, April 29, 2005.

^* This work was supported in part by grants from the National Institutes of Health (DK054354) and the Cystic Fibrosis Foundation (Kleyma03PO). 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.

Recipient of a postdoctoral fellowship award from the Pennsylvania-Delaware Affiliate of the American Heart Association. Supported by National Institutes of Health Grants DK061296 and DK066883.

^|| To whom correspondence should be addressed: Renal-Electrolyte Division, Dept. of Medicine, University of Pittsburgh, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-647-3121; Fax: 412-648-9166; E-mail: kleyman{at}pitt.edu.

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