Cytoplasmic terminus domains of Kir6.x confer different nucleotide-dependent gating on the ATP-sensitive K+ channel

1. In order to investigate the structural basis for the nucleotide-dependent gating of ATP-sensitive K+ channels (K-ATP), Kir6.1 (uKATP-1), Kir6.2 (BIR1) and chimeric channels were co-expressed with a common subtype of sulphonylurea receptor, SUR1, in COS7 cells. Representing the amino terminal domain-transmembrane domain-carboxyl-terminal domain of Kir6.1 as 1-1-1 and of Kir6.2 as 2-2-2, chimeric Kir6.x channels were constructed by swapping the amino and/or carboxyl terminal domains between Kir6.1 and Kir6.2 to give the chimeric x-l-x channels 1-1-2, 2-1-1 and 2-1-2, and the chimeric x-2-x channels 2-2-1, 1-2-2 and 1-2-1. 2. Inside-out patch clamp experiments revealed that both wild-type Kir6.1 and Kir6.2 formed inwardly rectifying K+ channels. Single-channel conductances were 36.3 and 66.1 pS, respectively Chimeric x-l-x channels, whose transmembrane domain was that of Kir6.1, showed similar ion-pore properties to wild-type Kir6.1. Likewise, chimeric x-2-x channels had similar ion-pore properties to wild-type Kir6.2. 3. Wild-type Kir6.1 and Kir6.2 possessed distinct gating properties towards intracellular nucleotides. The activity of Kir6.1 was entirely dependent on Mg2+ and nucleotide diphosphates (NDPs) such as UDP. In contrast, Kir6.2 was activated upon excision of patch membrane. When Kir6.2 underwent rundown, UDP reactivated the channel. 4. In order to eliminate UDP dependence from Kir6.1, it was necessary to replace both N- and C-termini; chimera 2-1-2 opened in UDP-free conditions. With Kir6.2, substitution of the N-terminus with that of Kir6.1 conferred UDP dependence on chimeras 1-2-2 and 1-2-1. Chimera 2-2-1 opened in UDP-free conditions, but UDP potentiated the channel activity by > 20-fold. 5. The kinetics of UDP-dependent activation were significantly different between Kir6.1. and Kir6.2. Kir6.1 maximally activated by UDP was sensitive to intracellular ATP, although its ATP sensitivity was significantly lower than that of Kir6.2 measured in identical conditions. The kinetics of UDP-dependent activation and ATP sensitivity could be transferred between Kir6.1 and Kir6.2 only when both N- and C-termini were replaced. We therefore concluded that nucleotide-dependent gating was regulated by the N- and C-terminal domains irrespective of the transmembrane domains.