title: betaL-betaM loop in the C-terminal domain of G protein-activated inwardly rectifying K(+) channels is important for G(betagamma) subunit activation.
abstract: The activity of G protein-activated inwardly rectifying K(+) channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although G(betagamma) subunits are known to bind the N- and C-termini of GIRK channels, the mechanism underlying G(betagamma) activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for G(betagamma) binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2(L344E) and GIRK2(G347H), that exhibited decreased carbachol-activated currents but significantly enhanced basal currents with coexpression of G(betagamma) subunits. Combining the two mutations (GIRK2(EH)) led to a more severe reduction in carbachol-activated and G(betagamma)-stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2(L344E) and GIRK2(EH) also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that G(betagamma)-impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced G(betagamma) activation. In vitro G(betagamma) binding assays revealed an approximately 60% decrease in G(betagamma) binding to the C-terminal domain of GIRK2(L344E) but no statistical change with GIRK2(EH) or GIRK2(G347H), though both mutants exhibited G(betagamma)-impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in G(betagamma) activation of GIRK2 channels. Based on the 1.8 A structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the betaL-betaM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which G(betagamma) alters the interaction between the betaL-betaM loop and the N-terminal domain.
authors.affiliation: Peptide Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Finley
authors.forename: Christine
authors.initials: C
authors.lastname: Arrabit
authors.forename: Catherine
authors.initials: C
authors.lastname: Fowler
authors.forename: Ka Fai
authors.initials: KF
authors.lastname: Suen
authors.forename: Paul A
authors.initials: PA
authors.lastname: Slesinger
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D005998:Glycine; D006639:Histidine; D006801:Humans; D007930:Leucine; D009865:Oocytes; D017354:Point Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014981:Xenopus
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D006639:Histidine; D007930:Leucine; D005998:Glycine