Subunit stoichiometry of a core conduction element in a cloned epithelial amiloride-sensitive Na+ channel

Biophys J. 1998 Nov;75(5):2292-301. doi: 10.1016/S0006-3495(98)77673-8.

Abstract

The molecular composition of a core conduction element formed by the alpha-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 alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amiloride / pharmacology*
  • Animals
  • Cloning, Molecular
  • Electrophysiology
  • Kinetics
  • Lipid Bilayers / metabolism
  • Protein Binding / physiology
  • Proteolipids / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Deletion / genetics
  • Sodium Channels / chemistry*
  • Sodium Channels / genetics
  • Xenopus

Substances

  • Lipid Bilayers
  • Proteolipids
  • Recombinant Proteins
  • Sodium Channels
  • proteoliposomes
  • Amiloride