title: Intrinsic voltage dependence of the epithelial Na+ channel is masked by a conserved transmembrane domain tryptophan.
abstract: Tryptophan residues critical to function are frequently located at the lipid-water interface of transmembrane domains. All members of the epithelial Na+ channel (ENaC)/Degenerin (Deg) channel superfamily contain an absolutely conserved Trp at the base of their first transmembrane domain. Here, we test the importance of this conserved Trp to ENaC/Deg function. Targeted substitution of this Trp in mouse ENaC and rat ASIC subunits decrease channel activity. Differential substitution with distinct amino acids in alpha-mENaC shows that it is loss of this critical Trp rather than introduction of residues having novel properties that changes channel activity. Surprisingly, Trp substitution unmasks voltage sensitivity. Mutant ENaC has increased steady-state activity at hyperpolarizing compared with depolarizing potentials associated with transient activation and deactivation times, respectively. The times of activation and deactivation change 1 ms/mV in a linear manner with rising and decreasing slopes, respectively. Increases in macroscopic currents at hyperpolarizing potentials results from a voltage-dependent increase in open probability. Voltage sensitivity is not influenced by divalent cations; however, it is Na+-dependent with a 63-mV decrease in voltage required to reach half-maximal activity per log increase in [Na+]. Mutant channels are particularly sensitive to intracellular [Na+] for removing this sodium abolishes voltage dependence. We conclude that the conserved Trp at the base of TM1 in ENaC/Deg channels protects against voltage by masking an inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.
authors.affiliation: Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
authors.forename: Oleh
authors.initials: O
authors.lastname: Pochynyuk
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Kucher
authors.forename: Nina
authors.initials: N
authors.lastname: Boiko
authors.forename: Elena
authors.initials: E
authors.lastname: Mironova
authors.forename: Alexander
authors.initials: A
authors.lastname: Staruschenko
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Karpushev
authors.forename: Qiusheng
authors.initials: Q
authors.lastname: Tong
authors.forename: Eunan
authors.initials: E
authors.lastname: Hendron
authors.forename: James
authors.initials: J
authors.lastname: Stockand
mesh_terms: D062565:Acid Sensing Ion Channels; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D062626:Degenerin Sodium Channels; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D051379:Mice; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
chemical_list: D062565:Acid Sensing Ion Channels; C568425:Asic2 protein, rat; D062626:Degenerin Sodium Channels; D053503:Epithelial Sodium Channels; D009419:Nerve Tissue Proteins; D015222:Sodium Channels; D014364:Tryptophan