Permeability Properties of Enac Selectivity Filter Mutants

Abstract

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.

The Extracellular Domain Determines the Kinetics of Desensitization in Acid-sensitive Ion Channel 1

“…These functional results together with the highly conserved sequence of M2 support the view that M2 forms the ion pathway (17,18). Although the region preceding M1 has also been implicated in ion permeation (11), we found little correlation of the poorly conserved amino termini with the pore properties.…”

Altered Na + transport after an intracellular α-subunit deletion reveals strict external sequential release of Na + from the Na/K pump

“…These findings are most easily explained by gua ϩ transiting the pathway through the exclusive Na ϩ site as proposed by Li et al (21). This novel effect of gua ϩ (mean geometric diameter, 4.4 Å) (31) sets minimal dimensions to the access channel normally transited by the first Na ϩ ion released from the exclusive site, indicating that Na ϩ may move in a hydrated form similar to proposals for Na ϩ permeation through VNaC (25). Although our data suggest that this pathway cannot accommodate NMG ϩ (mean diameter 7.3 Å) (32), consistent with the inability of NMG ϩ to inhibit the pump in a voltagedependent manner, we cannot be sure whether this access channel is narrower than the pathway opened by palytoxin on the Na/K-ATPase (33).…”

“…Guanidinium ϩ (gua ϩ ) ions compete with Na i ϩ for binding to the Na/K pump (23) and have been shown to permeate voltagedependent Na ϩ channels [VNaC (24), but not epithelial Na ϩ channels (25)]. To test whether gua ϩ is a Na ϩ -surrogate and can sustain inward currents through truncated pumps, we measured ouabain-sensitive currents with 120 mM gua o ϩ (Fig.…”

Outlines of the pore in open and closed conformations describe the gating mechanism of ASIC1

“…6c,d). Such a significant change in the geometry of the selectivity filter is an exception in the field of ion channels in particularly, because molecular sieving has been proposed as the basis for selecting Na + over K + in channels of the ENaC/Degenerins family [16][17][18] . It is unexpected that a channel that may rely on pore size to select the passing ions is also one that exhibits major changes in the diameter of the selectivity filter during gating.…”