A Single Point Mutation in the Distal C-Terminal of the Pore Forming Kir6.1 Subunit Modifies ATP-Sensitive Potassium (KATP) Channel Regulation

Abstract

KATP channels are hetero-octamers composed of two subunits; a pore forming Kir6 and a sulphonylurea receptor (SUR). SUR subunits regulate KATP channel gating allosterically, in response to nucleotides and pharmacological agents.We have previously shown that residue D323 of Kir6.2 is central to interaction with the nucleotide-binding fold 2 of SUR2A (Rubaiy et al, Biophysical J. 2011: 100; P432a). The aim of this study was to assess whether E332, the corresponding residue in Kir6.1, plays an equally important role in Kir6.1/SUR2A complexes. Wild type Kir6.1/SUR2A channels expressed in HEK293 cells required activation by potassium channel opener pinacidil (EC50 = 43.90 ± 1.28 µM), in the presence of UDP (10 mM) before passing current. Introduction of the single point mutation E332K into full length Kir6.1 caused constitutive opening of Kir6.1/SUR2A channels in the absence of pinacidil. Reinstatement of putative inter-subunit salt bridges by expressing Kir6.1-E332K with charge reversal mutants SUR2A-Q1336E or SUR2A-K1322D failed to restore regulated opening. Channels containing the Kir6.1E332K mutant were also insensitive to block by high concentrations of glibenclamide (100 µM). However, co-expression of Kir6.1-E332K with SUR2A-K1322D restored glibenclamide sensitivity to wild type levels ((IC50 = 9.12±1.12 nM), p < 0.15 versus wild type Kir6.1/SUR2A channel (IC50 = 6.14±1.13 nM). Together, these data suggest a key functional role for inter-subunit salt bridges involving Kir6.1-E332K. Constitutive channel opening on mutation of this residue suggests that Kir6.1E332 and salt bridge(s) formed between it and the SUR subunit are crucial for stabilizing closed states of Kir6.1-containing KATP channels.