Abstract
Removal of external K+ ions abolishes not only inward currents but also outward currents through the inwardly rectifying K+ channel which determines the resting potential. This suggests that external K+ ions are essential for the channel activation. To investigate how extracellular K+ ions act the channel, we made single points mutants in which one of negative charged amino acids in the extracellular loops of the murine inwardly rectifying K+ channel (Kir2.1) was neutralized. cDNA was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 24-72 h. COS-1 cells transfected with D112N did not show inward rectification under whole-cell recording in the normal Tyrode solution. Cells transfected with tandem tetramers with one wild-type (WT) and three D112N mutant subunits (WT-(D112N)3) also did show inward rectification, while cells transfected with tandem tetramers with two wild-type and two D112N subunits (WT2-(D112N)2) showed inward rectification. Channels from WT2-(D112N)2 had the single-channel conductance similar to that of wild-type channels. It is suggested that two negative charges at the D112 site may be required for K+ ions to bind to and activate the channel. [Jpn J Physiol 54 Suppl:S133 (2004)]
