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ATP-sensitive K(+)-channels link metabolism and excitability in neurons, myocytes, and pancreatic islets. Mutations in the pore-forming subunit (Kir6.2; KCNJ11) cause neonatal diabetes, developmental delay, and epilepsy by decreasing sensitivity to ATP inhibition and suppressing electrical activity. Mutations of residue G53 underlie both mild (G53R,S) and severe (G53D) forms of the disease. All examined substitutions (G53D,R,S,A,C,F) reduced ATP-sensitivity, indicating an intolerance of any amino acid other than glycine. Surprisingly, each mutation reduces ATP affinity, rather than intrinsic gating, although structural modeling places G53 at a significant distance from the ATP-binding pocket. We propose that glycine is required in this location for flexibility of the distal N-terminus, and for an induced fit of ATP at the binding site. Consistent with this hypothesis, glycine substitution of the adjacent residue (Q52G) partially rescues ATP affinity of reconstituted Q52G/G53D channels. The results reveal an important feature of the noncanonical ATP-sensing mechanism of K(ATP) channels.

The ROMK1 (Kir 1.1a) channel is formed by a tetrameric complex of subunits, each characterized by cytoplasmic N- and C-termini and a core region of two transmembrane helices flanking a pore-forming segment. To delineate the general regions mediating the assembly of ROMK1 subunits we constructed epitope-tagged N-terminal, C-terminal, and transmembrane segment deletion mutants. Nonfunctional subunits with N-terminal, core region, and C-terminal deletions had dominant negative effects when coexpressed with wild-type ROMK1 subunits in Xenopus oocytes. In contrast, coexpression of these nonfunctional subunits with Kv 2.1 (DRK1) did not suppress Kv 2.1 currents in control oocytes. Interactions between epitope-tagged mutant and wild-type ROMK1 subunits were studied in parallel by immunoprecipitating [35S]-labeled oocyte membrane proteins. Complexes containing both wild-type and mutant subunits that retained H5, M2, and C-terminal regions were coimmunoprecipitated to a greater extent than complexes consisting of wild-type and mutant subunits with core region and/or C-terminal deletions. The present findings are consistent with the hypothesis that multiple interaction sites located in the core region and cytoplasmic termini of ROMK1 subunits mediate homomultimeric assembly.