Regulation of a cloned epithelial Na+ channel by its beta- and gamma-subunits.
Using the Xenopus oocyte expression system, we examined the mechanisms by which the beta- and gamma-subunits of an epithelial Na+ channel (ENaC) regulate alpha-subunit channel activity and the mechanisms by which beta-subunit truncations cause ENaC activation. Expression of alpha-ENaC alone produced small amiloride-sensitive currents (-43 +/- 10 nA, n = 7). These currents increased > 30-fold with the coexpression of beta- and gamma-ENaC to -1,476 +/- 254 nA (n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the alpha-subunit of ENaC. Truncation of the last 75 amino acids of the beta-subunit COOH terminus, as found in the original pedigree of individuals with Liddle's syndrome, caused a 4.4-fold (n = 17) increase of the amiloride-sensitive currents compared with wild-type alpha beta gamma-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human beta-ENaC significantly reduced the amiloride-sensitive currents by 40-50%. These observations suggest a tonic inhibitory role on the channel's open probability (Po) by the COOH terminus of beta-ENaC. We conclude that the changes of current observed with coexpression of the beta- and gamma-subunits or those observed with beta-subunit truncation are likely the result of changes of channel density in combination with large changes of Po.
Awayda
M S
MS
Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Tousson
A
A
Benos
D J
DJ
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D003001:Cloning, Molecular; D006801:Humans; D006973:Hypertension / Q000235:genetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D017384:Sequence Deletion; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D013577:Syndrome; D013997:Time Factors; D014981:Xenopus
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D000584:Amiloride