title: The novel diazoxide analog 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide is a selective Kir6.2/SUR1 channel opener.
abstract: ATP-sensitive K(+) (K(ATP)) channels are activated by a diverse group of compounds known as potassium channel openers (PCOs). Here, we report functional studies of the Kir6.2/SUR1 Selective PCO 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216). We recorded cloned K(ATP) channel currents from inside-out patches excised from Xenopus laevis oocytes heterologously expressing Kir6.2/SUR1, Kir6.2/SUR2A, or Kir6.2/SUR2B, corresponding to the beta-cell, cardiac, and smooth muscle types of the K(ATP) channel. NNC 55-9216 reversibly activated Kir6.2/SUR1 currents (EC(50) = 16 micromol/l). This activation was dependent on intracellular MgATP and was abolished by mutation of a single residue in the Walker A motifs of either nucleotide-binding domain of SUR1. The drug had no effect on Kir6.2/SUR2A or Kir6.2/SUR2B currents. We therefore used chimeras of SUR1 and SUR2A to identify regions of SUR1 involved in the response to NNC 55-9216. Activation was completely abolished and significantly reduced by swapping transmembrane domains 8-11. The reverse chimera consisting of SUR2A with transmembrane domains 8-11 and NBD2 consisting SUR1 was activated by NNC 55-9216, indicating that these SUR1 regions are important for drug activation. [(3)H]glibenclamide binding to membranes from HEK293 cells transfected with SUR1 was displaced by NNC 55-9216 (IC(50) = 105 micromol/l), and this effect was impaired when NBD2 of SUR1 was replaced by that of SUR2A. These results suggest NNC 55-9216 is a SUR1-selective PCO that requires structural determinants, which differ from those needed for activation of the K(ATP) channel by pinacidil and cromakalim. The high selectivity of NNC 55-9216 may prove to be useful for studies of the molecular mechanism of PCO action.
authors.affiliation: Research & Development, Novo Nordisk, Malov, Denmark.
authors.forename: Michael
authors.initials: M
authors.lastname: Dabrowski
authors.forename: Frances M
authors.initials: FM
authors.lastname: Ashcroft
authors.forename: Rebecca
authors.initials: R
authors.lastname: Ashfield
authors.forename: Philippe
authors.initials: P
authors.lastname: Lebrun
authors.forename: Bernard
authors.initials: B
authors.lastname: Pirotte
authors.forename: Jan
authors.initials: J
authors.lastname: Egebjerg
authors.forename: John
authors.initials: J
authors.lastname: Bondo Hansen
authors.forename: Philip
authors.initials: P
authors.lastname: Wahl
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001581:Benzothiadiazines; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003981:Diazoxide / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004553:Electric Conductivity; D015870:Gene Expression; D005905:Glyburide / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects*; D051379:Mice; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000187:drug effects* / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins; D014162:Transfection; D014316:Tritium; D014982:Xenopus laevis
chemical_list: C457103:3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide; D001581:Benzothiadiazines; D007004:Hypoglycemic Agents; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D014316:Tritium; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D003981:Diazoxide; D005905:Glyburide