Nucleotide modulation of pinacidil stimulation of the cloned K(ATP) channel Kir6.2/SUR2A.
ATP-sensitive K(+) channels are the target for K(+) channel openers such as pinacidil. These channels are formed from pore-forming Kir6. 2 and regulatory sulfonylurea receptor (SUR) subunits. Pinacidil activates channels containing SUR2A (heart, skeletal muscle), but not those containing SUR1 (beta cells). Surprisingly, binding of the pinacidil analog [(3)H]P1075 is dependent on added nucleotides, yet in electrophysiological studies, pinacidil is effective in the absence of intracellular nucleotides. To determine the reason for this anomaly, we examined the functional interactions between pinacidil (or P1075) and nucleotides by expressing cloned Kir6. 2/SUR2A channels in Xenopus laevis oocytes. Both pinacidil and P1075 activated macroscopic Kir6.2/SUR2A currents in the absence of added nucleotide, but the presence of intracellular ATP or ADP slowed the off-rate of the response. Mutation of the Walker A lysine in a single nucleotide binding domain (NBD) of SUR2A (K707A in NBD1, K1348A in NBD2), abolished this action of nucleotide. The K1348A mutation prevented stimulation by MgADP but had little effect on the amplitude of the pinacidil response. In contrast, Kir6.2/SUR2A-K707A currents were activated by MgADP, but only responded to pinacidil in the presence of Mg-nucleotide. Off-rates in the absence (or presence) of nucleotide were slower for the pinacidil analog P1075 than for pinacidil, consistent with the higher affinity of P1075. We suggest that slowing of P1075 dissociation by nucleotide enables binding to be detected.
Gribble
F M
FM
University Laboratory of Physiology, Oxford, United Kingdom.
Reimann
F
F
Ashfield
R
R
Ashcroft
F M
FM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology*; D001665:Binding Sites; D006146:Guanidines / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D020110:Pinacidil / Q000494:pharmacology*; D015221:Potassium Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011487:Protein Conformation; D011725:Pyridines / Q000494:pharmacology; D014316:Tritium; D014665:Vasodilator Agents / Q000494:pharmacology; D014982:Xenopus laevis
D000959:Antihypertensive Agents; D006146:Guanidines; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011725:Pyridines; D014665:Vasodilator Agents; D014316:Tritium; C073896:N-cyano-N'-(1,1-dimethylpropyl)-N''-(3-pyridinyl)guanidine; D000244:Adenosine Diphosphate; D020110:Pinacidil; D000255:Adenosine Triphosphate