INTRACELLULAR NUCLEOTIDE-MEDIATED GATING OF SUR KIR6.0 COMPLEX POTASSIUM CHANNELS EXPRESSED IN A MAMMALIAN-CELL LINE AND ITS MODIFICATION BY PINACIDIL/

1. We have examined the properties of intracellular nucleotide-mediate d gating of K+ channel constructs composed of the sulphonylurea recept or 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6. 2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously e xpressed in human embryonic kidney (HEK) 293T cells. In the cell-attac hed form, both types of K+ channel were activated by pinacidil. 2. In inside-out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneousl y and were inhibited by intracellular ATP (ATP(i)). Pinacidil attenuat ed the ATP(i)-mediated channel inhibition in a concentration-dependent manner. In contrast, the SUR2B/Kir6.1. channels required intracellula r nucleoside di- or tri-, but not mono-, phosphates for opening. The p otency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir 6.1 channels was enhanced by pinacidil. 3. In the presence of pinacidi l, adenine and guanine, but not uracil, nucleotides exhibited bell-sha ped concentration-dependent activating effects on SUR2B/Kir6.1 channel s. This was due to channel inhibition caused by adenine and guanine nu cleotides, which was unaffected by pinacidil. 4. From power density sp ectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide-independent fast c hannel gate and a nucleotide-dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the p otency of nucleotide action on the slow gate. 5. We conclude that Kir6 .0 subunits play a crucial role in the nucleotide-mediated gating of S UR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.