title: Gating of the ATP-sensitive K+ channel by a pore-lining phenylalanine residue.
abstract: ATP-sensitive K(+) (K(ATP)) channels are gated by intracellular ATP, proton and phospholipids. The pore-forming Kir6.2 subunit has all essential machineries for channel gating by these ligands. It is known that channel gating involves the inner helix bundle of crossing in which a phenylalanine residue (Phe168) is found in the TM2 at the narrowest region of the ion-conduction pathway in the Kir6.2. Here we present evidence that Phe168-Kir6.2 functions as an ATP- and proton-activated gate via steric hindrance and hydrophobic interactions. Site-specific mutations of Phe168 to a small amino acid resulted in losses of the ATP- and proton-dependent gating, whereas the channel gating was well maintained after mutation to a bulky tryptophan, supporting the steric hindrance effect. The steric hindrance effect, though necessary, was insufficient for the gating, as mutating Phe168 to a bulky hydrophilic residue severely compromised the channel gating. Single-channel kinetics of the F168W mutant resembled the wild-type channel. Small residues increased P(open), and displayed long-lasting closures and long-lasting openings. Kinetic modeling showed that these resulted from stabilization of the channel to open and long-lived closed states, suggesting that a bulky and hydrophobic residue may lower the energy barrier for the switch between channel openings and closures. Thus, it is likely that the Phe168 acts as not only a steric hindrance gate but also potentially a facilitator of gating transitions in the Kir6.2 channel.
authors.affiliation: Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, GA 30302-4010, USA.
authors.forename: Asheebo
authors.initials: A
authors.lastname: Rojas
authors.forename: Jianping
authors.initials: J
authors.lastname: Wu
authors.forename: Runping
authors.initials: R
authors.lastname: Wang
authors.forename: Chun
authors.initials: C
authors.lastname: Jiang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry; D014982:Xenopus laevis; D000099161:Kcnj11 Channel
chemical_list: D000255:Adenosine Triphosphate; D010649:Phenylalanine; D024661:Potassium Channels, Inwardly Rectifying; D014364:Tryptophan; D000099161:Kcnj11 Channel