抄録
Removal of external K+ ions abolishes not only inward currents but also outward currents through inwardly rectifying K+ channels which determine the resting potential. Negatively charged amino acid residues located near the external entrance are expected to attract and stabilize K+ ions to modulate channel gating or block. To understand the significance of negative charges (D112, D114, E125, D152 and E153) in the extracellular loops of the inwardly rectifying K+ (Kir2.1) channel, single or double point mutants were constructed and transfected into COS-1 and HEK293 cells. Each single point mutant expressed functional channels. Cells transfected with the D152N/E153Q construct did not show any inwardly rectifying K+ currents, although fluorescence images confirmed that the channel proteins produced by the D152N/E153Q construct were transported to the cell surface. While a tandem tetramer with one D152N subunit and three double mutant subunits, D152N-(D152N/E153Q)3, did not express functional channels, a tandem tetramer with one E153Q subunit and three double mutant subunits, E153Q-(D152N/E153Q)3, and that with two D152N subunits and two double mutant subunits, (D152N)2-(D152N/E153Q)2 expressed Kir2.1 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 K+ ions may be stabilized at D152 and/or E153 site and K+ stabilization at this site sets the channel to function. [J Physiol Sci. 2007;57 Suppl:S222]
