The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.
allostery, binding, diabetes, gating, human, ligand, metabolism, molecular biophysics, structural biology, Adenosine Triphosphate, Binding Sites, Enzyme Activation, HEK293 Cells, Humans, KATP Channels, Kinetics, Magnesium, Mutagenesis, Site-Directed, Potassium Channels, Inwardly Rectifying, Protein Binding, Protein Conformation, Sulfonylurea Receptors