Conformational changes in Kir2.1 channels during NH4+-induced inactivation

We have shown previously that NH4+ 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 NH4+-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 NH4+-induced inactivation. In addition, we examined whether the mutation of amino acids located at various parts of a Kir2.1 channel influences the N-H-4(+)-induced inactivation. Kir2.1 channels were expressed in Xenopus oocytes and studied using patch-clamp techniques. The gating of the NH4+-induced inactivation was affected by mutation of several amino acids located at various regions of the Mr2.1 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 NH4+-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 NH4+-induced inactivation in the Kir2.1 channel.