TY - JOUR
T1 - Intrinsic voltage dependence of the epithelial Na+ channel is masked by a conserved transmembrane domain tryptophan
AU - Pochynyuk, Oleh
AU - Kucher, Volodymyr
AU - Boiko, Nina
AU - Mironova, Elena
AU - Staruschenko, Alexander
AU - Karpushev, Alexey V.
AU - Tong, Qiusheng
AU - Hendron, Eunan
AU - Stockand, James
PY - 2009/9/18
Y1 - 2009/9/18
N2 - 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 α-mENaCshows 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.Weconclude that the conserved Trp at the base of TM1inENaC/Degchannels protects against voltagebymaskingan inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.
AB - 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 α-mENaCshows 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.Weconclude that the conserved Trp at the base of TM1inENaC/Degchannels protects against voltagebymaskingan inhibitory allosteric or pore block mechanism, which decreases activity in response to intracellular Na+.
UR - http://www.scopus.com/inward/record.url?scp=70350023553&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=70350023553&partnerID=8YFLogxK
U2 - 10.1074/jbc.M109.015917
DO - 10.1074/jbc.M109.015917
M3 - Article
C2 - 19620245
AN - SCOPUS:70350023553
VL - 284
SP - 25512
EP - 25521
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 38
ER -