Activation mechanism of ATP-sensitive K⁺ channels explored with real-time nucleotide binding.

Abstract

The response of ATP-sensitive K⁺ channels (K_ATP) 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_ATP 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 <i>EC₅₀</i> 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 K_ATP activation by the same amount.