Intracellular nucleotide‐mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil

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Abstract
1 We have examined the properties of intracellular nucleotide‐mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell‐attached form, both types of K+ channel were activated by pinacidil. 2 In inside‐out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi‐mediated channel inhibition in a concentration‐dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di‐ or tri‐, but not mono‐, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil. 3 In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell‐shaped concentration‐dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil. 4 From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide‐independent fast channel gate and a nucleotide‐dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate. 5 We conclude that Kir6.0 subunits play a crucial role in the nucleotide‐mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.

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