The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide.
The ATP-sensitive K-channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg-ADP, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (W(A)) motifs of the first (K719A) and second (K1384M) nucleotide-binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the W(A) lysine of NBD1 (but not NBD2) is essential for activation of K-ATP currents by diazoxide. The potentiatory effects of Mg-ADP required the presence of the W(A) lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild-type currents. Metabolic inhibition led to activation of wild-type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K-ATP channel activity.
Gribble
F M
FM
University Laboratory of Physiology, Oxford, UK.
Tucker
S J
SJ
Ashcroft
F M
FM
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D003001:Cloning, Molecular; D003981:Diazoxide / Q000494:pharmacology*; D005260:Female; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D019810:Sodium Azide; D014981:Xenopus
D000963:Antimetabolites; D001386:Azides; D007328:Insulin; D015221:Potassium Channels; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D008274:Magnesium; D003981:Diazoxide