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<jats:title>Abstract</jats:title><jats:p>The response of ATP-sensitive K<jats:sup>+</jats:sup> channels (K<jats:sub>ATP</jats:sub>) 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 K<jats:sub>ATP</jats:sub> in channel gating. Binding to NBS2 was Mg<jats:sup>2+</jats:sup>-independent, but Mg was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC<jats:sub>50</jats:sub> for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg<jats:sup>2+</jats:sup>, 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 K<jats:sub>ATP</jats:sub> activation by the same amount.</jats:p>

Original publication




Journal article


Cold Spring Harbor Laboratory

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