An Intramolecular Interaction Controls a Rate-Limiting Step in ATP-Dependent Gating of Kir6.2 Channels

Abstract

ATP-sensitive potassium (KATP) channels are complexes of inwardly-rectifying Kir channels (Kir6.x) and sulfonylurea receptors (SUR). These two subunits work together to encode sensitivity to intracellular adenine nucleotides (ATP and ADP) and other regulatory ligands including PIP2. We investigated the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels rapidly re-open after washout of ATP, with a time constant of ∼60 ms. Extending similar kinetic measurements to mutants around the bundle crossing revealed modest effects on gating kinetics despite significant changes in ATP sensitivity. However, we identified a pair of highly conserved neighboring amino acids (Trp68, Lys170) that controls the rate of channel recovery and inhibition by ATP. Mutations of Trp68 or Lys170 have the paradoxical effect of dramatically slowing the kinetics of channel opening, while increasing channel open probability. Formalization of the transition state effects of these residues using phi-value analysis revealed a consistent steep negative slope. This finding implies that these residues play a specific role in lowering the transition state energy barrier between open and closed channel states. The demonstration of a negative phi-value is unique for an ion channel gating process, and diverges from interpretations that use phi-values to place specific residues in the spatial and temporal progression through the transition energy landscape. Using nonsense suppression for unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68, potentially required to localize the e-amine of Lys170 in the PIP2 binding site. Our findings identify a discrete pair of residues with an essential role for controlling gating kinetics of Kir channels.