Negatively charged residues located near the external entrance are required for the Kir2.1 channel to function

To understand the significance of negative charges in the extracellular loops of the inwardly rectifying K+ (Kir2.1) channel, single-point mutants (D112N, D114N, E125Q, D152E, D152K, D152N, E153D, E153K, and E153Q) and double-point mutants (D112N/D114N and D152N/E153Q) were constructed and transfected into COS-1 and HEK293 cells. All single-point mutants, except D152K, and D112N/D114N expressed functional channels. Cells transfected with the D152K and D152N/E153Q constructs did not show any inwardly rectifying K+ currents, although fluorescence images confirmed that the channel proteins produced by D152K and D152N/E153Q were transported to the cell surface. While a tandem tetramer with one D152N subunit and three D152N/E153Q subunits, D152N–(D152N/E153Q)3, did not express functional channels, a tandem tetramer with one E153Q subunit and three D152N/E153Q subunits, E153Q–(D152N/E153Q)3, and that with two D152N subunits and two D152N/E153Q subunits, (D152N)2–(D152N/E153Q)2, expressed channels having similar conductance and kinetics of single-channel currents to the wild-type channels. These results suggest that one negative charge of D152 or two negative charges of E153 are required for Kir2.1 channels to function. It is suggested that the contribution by D152 and E153 to the electronegative extracellular pore entrance is critical for the channel to function properly.