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J Biol Chem, Vol. 275, Issue 12, 8572-8581, March 24, 2000
From the Departments of Medicine and Physiology, School of
Medicine, University of Pennsylvania and the Veteran Affairs Medical
Center, Philadelphia, Pennsylvania 19104
The epithelial sodium channel (ENaC) is composed
of three homologous subunits termed
Characterization of the Selectivity Filter of the Epithelial
Sodium Channel*
,,
, 
, and 
. Previous studies
suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse
ENaC by systematically mutating all 24 amino acids within this putative
pore region of the -subunit to cysteine and co-expressing these
mutants with wild type - and -subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these
mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the
functional changes associated with point mutations. An amino-terminal
domain (-Val569--Gly579) showed minimal
changes in cation selectivity or amiloride sensitivity following
cysteine substitution. In contrast, cysteine substitutions within the
carboxyl-terminal domain
(-Ser580--Ser592) resulted in
significant changes in cation selectivity and moderately altered
amiloride sensitivity. The mutant channels containing G587C or
S589C were permeable to K+, and mutation of a GSS tract
(positions 587-589) to GYG resulted in a moderately
K+-selective channel. Our results suggest that the
C-terminal portion of the pore region within the -subunit
contributes to the selectivity filter of ENaC.
*
This work was supported by National Institutes of Health
Grant DK54354 and a grant from the Department of Veterans Affairs.The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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 Cystic
Fibrosis Foundation.
§
To whom correspondence should be addressed: Renal Division, 700 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6144. Tel.: 215-573-1848; Fax: 215-898-0189; E-mail:
kleyman@mail.med.upenn.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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