title: Forced gating motions by a substituted titratable side chain at the bundle crossing of a potassium channel.
abstract: Numerous inwardly rectifying potassium (Kir) channels possess an aromatic residue in the helix bundle crossing region, forming the narrowest pore constriction in crystal structures. However, the role of the Kir channel bundle crossing as a functional gate remains uncertain. We report a unique phenotype of Kir6.2 channels mutated to encode glutamate at this position (F168E). Despite a prediction of four glutamates in close proximity, Kir6.2(F168E) channels are predominantly closed at physiological pH, whereas alkalization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated at alkaline pH, forcing channel opening due to mutual repulsion of nearby negatively charged side chains. The potassium channel pore scaffold likely brings these glutamates close together, causing a significant pK(a) shift relative to the free side chain (as seen in the KcsA selectivity filter). Alkalization also shifts the apparent ATP sensitivity of the channel, indicating that forced motion of the bundle crossing is coupled to the ATP-binding site and may resemble conformational changes involved in wild-type Kir6.2 gating. The study demonstrates a novel mechanism for engineering extrinsic control of channel gating by pH and shows that conformational changes in the bundle crossing region are involved in ligand-dependent gating of Kir channels.
authors: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors: Anu
authors: A
authors: Khurana
authors: Evan S
authors: ES
authors: Shao
authors: Robin Y
authors: RY
authors: Kim
authors: Yury Y
authors: YY
authors: Vilin
authors: Xinyang
authors: X
authors: Huang
authors: Runying
authors: R
authors: Yang
authors: Harley T
authors: HT
authors: Kurata
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D051379:Mice; D020125:Mutation, Missense; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D014982:Xenopus laevis; D000099161:Kcnj11 Channel
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; D000099161:Kcnj11 Channel