Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K+ channels.
The cytoplasmic domain of inward rectifier K(+) (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent βD and βH strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K(+) currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit. Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.
Department of Pharmacology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan ; Center for Advanced Medical Engineering and Informatics, Osaka University, Suita, Osaka, Japan.
Atsushi
A
Inanobe
Atsushi
A
Nakagawa
Yoshihisa
Y
Kurachi
D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D002414:Cations, Monovalent; D004305:Dose-Response Relationship, Drug; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D015640:Ion Channel Gating; D017136:Ion Transport; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043585:Receptor, Muscarinic M2 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D002414:Cations, Monovalent; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C497069:Kcnj6 protein, mouse; D021122:Protein Subunits; D043585:Receptor, Muscarinic M2; D011994:Recombinant Proteins; D018698:Glutamic Acid; D008715:Methionine; D000109:Acetylcholine; D011188:Potassium