An amino acid triplet in the NH2 terminus of rat ROMK1 determines interaction with SUR2B

ATP-regulated (K-ATP) channels are formed by an inward rectifier pore-formi ng subunit (Kir) and a sulfonylurea (glibenclamide)-binding protein, a memb er of the ATP binding cassette family (sulfonylurea receptor (SUR) or cysti c fibrosis transmembrane conductance regulator). The latter is required to confer glibenclamide sensitivity to KATP channels. In the mammalian kidney ROMK1-3 are components of K-ATP channels that mediate K+ secretion into uri ne. ROMK1 and ROMK3 splice variants share the core polypeptide of ROMK2 but also have distinct NH2-terminal extensions of 19 and 26 amino acids, respe ctively. The SUR2B is also expressed in rat kidney tubules and may combine with Kir.1 to form renal K-ATP channels. Our previous studies showed that c o-expression of ROMK2, but not ROMK1 or ROMK3, with rat SUR2B in oocytes ge nerated glibenclamide-sensitive K+ currents. These data suggest that the NH 2-terminal extensions in both ROMK1 and ROMK3 block ROMK-SUR2B interaction. Seven amino acids in the NH2-terminal extensions of ROMK1 and ROMK3 are id entical (amino acids 13-19 in ROMK1 and 20-26 in ROMK3) and may determine R OMK-SUR2B interaction. We constructed a series of hemagglutinin-tagged ROM] KI NH2-terminal deletion and substitution mutants and examined glibenclamid e-sensitive K+ currents in oocytes when co-expressed with SUR2B. These stud ies identified an amino acid triplet "IRA" within the conserved segment in the NH2 terminus of ROMK1 and ROMK3 that blocks the ability of SUR2B to con fer glibenclamide sensitivity to the expressed K+ currents. The position of this triplet in the ROMK1 NH2-terminal extension is also important for the ROMK-SUR2B interactions. In vitro co-translation and immunoprecipitation s tudies with hemagglutinin-tagged ROMK mutants and SUR2B indicted that direc t interaction between these two proteins is required for glibenclamide sens itivity of induced K+ currents in oocytes. These results suggest that the I RA triplet in the NH2-terminal extensions of both ROMK1 and ROMK3 plays a k ey role in subunit assembly of the renal secretary K-ATP channel.