Subunit Stoichiometry of a Core Conduction Element in a Cloned Epithelial Amiloride-Sensitive Na+ Channel


The molecular composition of a core conduction element formed by the α-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated αΔN-rENaC that displays accelerated kinetics (τo = 32 ± 13 ms, τc = 42 ± 11 ms), as compared with the WT channel (τc1 = 18 ± 8 ms, τc2 = 252 ± 31 ms, and τo = 157 ± 43 ms); and 2) WT and an amiloride binding mutant, αΔ278–283-rENaC. The channels that formed in a αWT:αΔN mixture fell into two groups: one with τo and τc that corresponded to those exhibited by the αΔN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the αWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1αWT:1αΔ278–283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of α-subunits as a minimal model for the core conduction element in ENaCs.

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Biophysical Journal



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© 1998 The Biophysical Society.