A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels

作者：Zhang Roger S; Wright Jordan D; Pless Stephan A; Nunez John Jose; Kim Robin Y; Li Jenny B W; Yang Runying; Ahern Christopher A; Kurata Harley T^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2015, 290(25): 15450-15461.

DOI：10.1074/jbc.M114.631960

• diabetes
• gating
• ion channel
• kinetics
• potassium channel
• KATP channel
• PIP2
• ligand-dependent gating

Background: Kir6.2 potassium channels are regulated by ATP. Results: We measured Kir6.2 gating kinetics in response to rapid ATP concentration jumps. Mutations to Trp-68 and Lys-170 dramatically decelerate gating. Conclusion: Trp-68 and Lys-170 interact to form a cluster that enables rapid gating transitions. Significance: The Trp-68/Lys-170 cluster is highly conserved and may play a similar role in other Kir channels.
ATP-sensitive potassium (K-ATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of K-ATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of approximate to 60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using phi value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the E-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

• 出版日期2015-6-19