Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding.
ATP-sensitive potassium (K(ATP)) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K(+) channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved "signature sequence" (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 microM) to displace 8-azido-[(32)P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced K(ATP) current activation by 100 microM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-R50G co-expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.
Matsuo
Michinori
M
University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.
Dabrowski
Michael
M
Ueda
Kazumitsu
K
Ashcroft
Frances M
FM
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D005260:Female; D007003:Hypoglycemia / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D014161:Transduction, Genetic; D014981:Xenopus
D018528:ATP-Binding Cassette Transporters; D024661:Potassium Channels, Inwardly Rectifying; D000255:Adenosine Triphosphate