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-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.
Department of Pharmacology II, Faculty of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
E
E
Satoh
M
M
Yamada
C
C
Kondo
V P
VP
Repunte
Y
Y
Horio
T
T
Iijima
Y
Y
Kurachi
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D007668:Kidney / Q000737:chemistry / Q000166:cytology; D007700:Kinetics; D008322:Mammals; D006899:Mixed Function Oxygenases / Q000502:physiology*; D020110:Pinacidil / Q000494:pharmacology*; D015221:Potassium Channels / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D014530:Uridine Diphosphate / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology; D014665:Vasodilator Agents / Q000494:pharmacology*
D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D014665:Vasodilator Agents; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D020110:Pinacidil; D000255:Adenosine Triphosphate; D006899:Mixed Function Oxygenases; D014544:Uridine Triphosphate