Conformational changes in Kir2.1 channels during NH4+-induced inactivation.
We have shown previously that NH(4)(+) binding to the external pore of a Kir2.1 channel induces channel inactivation possibly through conformational changes. In this study, we performed further biophysical analyses of the NH(4)(+)-induced inactivation modeled by a refined kinetic scheme. Also, we investigated the conformational change hypothesis by examining whether the chemical modification of single-cysteine substitution of amino acids located at the internal pore alters the kinetics of the NH(4)(+)-induced inactivation. In addition, we examined whether the mutation of amino acids located at various parts of a Kir2.1 channel influences the NH(4)(+)-induced inactivation. Kir2.1 channels were expressed in Xenopus oocytes and studied using patch-clamp techniques. The gating of the NH(4)(+)-induced inactivation was affected by mutation of several amino acids located at various regions of the Kir2.1 channel. These results suggest that amino acids from different parts of a Kir2.1 channel are involved in the channel closure. Furthermore, internal chemical modification of several cysteine mutants resulted in the block of inward currents and changes in the on and off rate for the NH(4)(+)-induced inactivation, suggesting that the internal pore mouth is involved in the closure of a Kir2.1 channel. Taken together these results provide new evidence for conformational changes affecting the NH(4)(+)-induced inactivation in the Kir2.1 channel.
Chang
Hsueh-Kai
HK
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China.
Shieh
Ru-Chi
RC
D000818:Animals; D003545:Cysteine; D005260:Female; D007700:Kinetics; D009154:Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000187:drug effects; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D014981:Xenopus
D024661:Potassium Channels, Inwardly Rectifying; D000644:Quaternary Ammonium Compounds; D003545:Cysteine