TY - JOUR TI - Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding AU - Puljung, Michael AU - Vedovato, Natascia AU - Usher, Samuel AU - Ashcroft, Frances A2 - Kuriyan, John A2 - Swartz, Kenton Jon A2 - Swartz, Kenton Jon A2 - Shyng, Show-Ling A2 - Goldschen-Ohm, Marcel P VL - 8 PY - 2019 DA - 2019/02/21 SP - e41103 C1 - eLife 2019;8:e41103 DO - 10.7554/eLife.41103 UR - https://doi.org/10.7554/eLife.41103 AB - 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. KW - allostery KW - binding KW - gating KW - diabetes KW - ligand KW - metabolism JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -