In search of the structural basis for gating of amiloride-sensitive Na+ channels, kinetic properties of single homo and heterooligomeric ENaCs formed by the subunits with individual truncated cytoplasmic domains were studied in a cell-free planar lipid bilayer reconstitution system. Our results identify the N-terminus of the α-subunit as a major determinant of kinetic behavior of both homooligomeric and heterooligomeric ENaCs, although the carboxy- terminal domains of β, and γ-ENaC subunits play important role(s) in modulation of the kinetics of heterooligomeric channels. We also found that the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits amiloride-sensitive channels, at least in part, by modulating their gating. Comparison of these data suggests that the modulatory effects of the β- and γ-ENaC subunits, and of the CFTR, may involve the same, or closely related, mechanism(s); namely, 'locking' the heterooligomeric channels in their closed state. These mechanisms, however, do not completely override the gating mechanism of the α-channel.
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