The epithelial Na+ channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na+ absorption. The amiloride-sensitive ENaC is highly selective for Na+ and Li+ ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the αS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K+ ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted αS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position α589, indicating that αS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na+ and Li+. These findings demonstrate the critical role of the pore size at the αS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the αS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the αS589 side chain is oriented toward the subunit–subunit interface and that substitution of αS589 by larger residues increases the pore diameter by adding extra volume at the subunit–subunit interface.
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1 December 2001
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November 26 2001
Permeability Properties of Enac Selectivity Filter Mutants
Stephan Kellenberger,
Stephan Kellenberger
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
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Muriel Auberson,
Muriel Auberson
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
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Ivan Gautschi,
Ivan Gautschi
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
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Estelle Schneeberger,
Estelle Schneeberger
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
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Laurent Schild
Laurent Schild
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
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Stephan Kellenberger
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
Muriel Auberson
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
Ivan Gautschi
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
Estelle Schneeberger
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
Laurent Schild
aInstitut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
Abbreviations used in this paper: DEG, degenerin; DMA, dimethylammonium; ENaC, epithelial Na+ channel; ILi, amiloride-sensitive Li+ current; INa, amiloride-sensitive Na+ current; INH4, amiloride-sensitive NH4+ current; M2, second transmembrane domain; M2Ab, anti-FLAG M2 mouse mAb; pre-M2, segment NH2-terminal of M2; MA, methylammonium; MTSEA, aminoethyl methanethiosulfate; TriMA, trimethylammonium; wt, wild type.
Received:
May 10 2001
Revision Requested:
October 19 2001
Accepted:
October 22 2001
Online ISSN: 1540-7748
Print ISSN: 0022-1295
© 2001 The Rockefeller University Press
2001
The Rockefeller University Press
J Gen Physiol (2001) 118 (6): 679–692.
Article history
Received:
May 10 2001
Revision Requested:
October 19 2001
Accepted:
October 22 2001
Citation
Stephan Kellenberger, Muriel Auberson, Ivan Gautschi, Estelle Schneeberger, Laurent Schild; Permeability Properties of Enac Selectivity Filter Mutants. J Gen Physiol 1 December 2001; 118 (6): 679–692. doi: https://doi.org/10.1085/jgp.118.6.679
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