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-forming subunit (Kir) and a sulfonylurea (glibenclamide)-binding protein, a member of the ATP binding cassette family (sulfonylurea receptor (SUR) or cystic fibrosis transmembrane conductance regulator). The latter is required to confer glibenclamide sensitivity to K_ATP channels. In the mammalian kidney ROMK1-3 are components of K_ATP channels that mediate K ⁺ secretion into urine. ROMK1 and ROMK3 splice variants share the core polypeptide of ROMK2 but also have distinct NH₂-terminal extensions of 19 and 26 amino acids, respectively. 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 co-expression of ROMK2, but not ROMK1 or ROMK3, with rat SUR2B in oocytes generated glibenclamide-sensitive K⁺ currents. These data suggest that the NH₂-terminal extensions in both ROMK1 and ROMK3 block ROMK-SUR2B interaction. Seven amino acids in the NH₂-terminal extensions of ROMK1 and ROMK3 are identical (amino acids 13-19 in ROMK1 and 20-26 in ROMK3) and may determine ROMK-SUR2B interaction. We constructed a series of hemagglutinin-tagged ROMK1 NH₂-terminal deletion and substitution mutants and examined glibenclamide-sensitive K⁺ currents in oocytes when co-expressed with SUR2B. These studies 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 confer 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 studies with hemagglutinin-tagged ROMK mutants and SUR2B indicted that direct interaction between these two proteins is required for glibenclamide sensitivity of induced K⁺ currents in oocytes. These results suggest that the IRA triplet in the NH₂-terminal extensions of both ROMK1 and ROMK3 plays a key role in subunit assembly of the renal secretary K_ATP channel.