NEUTRALIZATION OF ASPARTATE RESIDUES IN THE MURINE INWARDLY RECTIFYING K+ CHANNEL IRK1 AFFECTS THE SUBSTATE BEHAVIOR IN MG2+ BLOCK

1. To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single-channel cur rents through inwardly rectifying K+ (JRK1) channels were studied. 2. cDNA encoding a functional murine IRK1 channel was transfected into CO X-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage damp experiments were done after 48-72 h. 3. Intracellula r Mg2+ at micromolar concentrations induced sublevels in the outward c urrent at one-third and two-thirds of the unitary amplitude seen in wi ld-type channels. Replacing Asp 172 with Asn (D172N) and Gin (D172Q) a bolished these sublevels, i.e. the channel showed only the fully open and fully blocked states. 4. Both mutations reduced the Mg2+ sensitivi ty of the channel at 2 mu M Mg2+. However, the Mg2+ sensitivity did no t differ significantly at higher concentrations (10 mu M) and voltages (+70 mV). 5. Channels expressed from D172E showed the sublevels, indi cating that a negative charge is indispensable to the substate behavio ur. 6. Channels from tandem tetramers of IRK1 with one and two D172N m utant subunits mainly showed sublevels with two-thirds amplitude, whil e those from tetramers with three D172N mutant subunits showed no subl evels. 7. These findings suggest that differences in Mg2+ binding patt erns lead to different conductive states in a single-barrelled channel .