NMR and fluorescence studies of drug binding to the first nucleotide binding domain of SUR2A.
ATP sensitive potassium (K(ATP)) channels are composed of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B) that surround the pore. SUR proteins are members of the ATP-binding cassette (ABC) superfamily of proteins. Binding of MgATP at the SUR nucleotide binding domains (NBDs) results in NBD dimerization, and hydrolysis of MgATP at the NBDs leads to channel opening. The SUR proteins also mediate interactions with K(ATP) channel openers (KCOs) that activate the channel, with KCO binding and/or activation involving residues in the transmembrane helices and cytoplasmic loops of the SUR proteins. Because the cytoplasmic loops make extensive interactions with the NBDs, we hypothesized that the NBDs may also be involved in KCO binding. Here, we report nuclear magnetic resonance (NMR) spectroscopy studies that demonstrate a specific interaction of the KCO pinacidil with the first nucleotide binding domain (NBD1) from SUR2A, the regulatory SUR protein in cardiac K(ATP) channels. Intrinsic tryptophan fluorescence titrations also demonstrate binding of pinacidil to SUR2A NBD1, and fluorescent nucleotide binding studies show that pinacidil binding increases the affinity of SUR2A NBD1 for ATP. In contrast, the KCO diazoxide does not interact with SUR2A NBD1 under the same conditions. NMR relaxation experiments and size exclusion chromatography indicate that SUR2A NBD1 is monomeric under the conditions used in drug binding studies. These studies identify additional binding sites for commonly used KCOs and provide a foundation for testing binding of drugs to the SUR NBDs.
Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, Ontario, Canada L5L 1C6.
Jorge P
JP
López-Alonso
Elvin D
ED
de Araujo
Voula
V
Kanelis
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003981:Diazoxide / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009711:Nucleotides / Q000378:metabolism; D020110:Pinacidil / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism* / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D011955:Receptors, Drug / Q000378:metabolism* / Q000502:physiology; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; C577509:Abcc8 protein, rat; D009711:Nucleotides; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D020110:Pinacidil; D000255:Adenosine Triphosphate; D003981:Diazoxide