Neutralization of aspartate residues in the murine inwardly rectifying K+channel IRK1 affects the substate behaviour in Mg2+block

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Abstract
1. To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single-channel currents through inwardly rectifying K+ (IRK1) channels were studied. 2. cDNA encoding a functional murine IRK1 channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 3. Intracellular Mg2+ at micromolar concentrations induced sublevels in the outward current at one-third and two-thirds of the unitary amplitude seen in wild-type channels. Replacing Asp 172 with Asn (D172N) and Gln (D172Q) abolished these sublevels, i.e. the channel showed only the fully open and fully blocked states. 4. Both mutations reduced the Mg2+ sensitivity of the channel at 2 microM Mg2+. However, the Mg2+ sensitivity did not differ significantly at higher concentrations (10 microM) and voltages (+70 mV). 5. Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behaviour. 6. Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two-thirds amplitude, while those from tetramers with three D172N mutant subunits showed no sublevels. 7. These findings suggest that differences in Mg2+ binding patterns lead to different conductive states in a single-barrelled channel.