title: Identification of regions that regulate the expression and activity of G protein-gated inward rectifier K+ channels in Xenopus oocytes.
abstract: 1. The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein-gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild-type subunits together with the m2 muscarinic receptor in Xenopus oocytes. 2. Co-expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1-Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane. 3. Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine-induced channel activity compared with the wild-type Kir3.2 homomeric channel. Cell-attached, single-channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings. 4. Individually substituting the N- and C-terminal tails of Kir3.1 for those of Kir3.2 showed that the C-terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N-terminal or core regions of this subunit. 5. The chimeric channel, 121, displayed a higher ratio of ACh-induced to basal activity than the Kir3.1-Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for G beta gamma. Our results suggest that substituting the Kir3.1 C-terminal tail for the Kir3.2 tail promotes the opening conformational change of the G beta gamma-bound channel. 6. The core and C-terminal regions of Kir3.1 independently conferred time dependence on voltage-dependent activation. The time constant (tau) was between 5 and 10 ms and varied little over the voltage range -60 to -120 mV.
authors.affiliation: Department of Pharmacology, University of Cambridge, UK.
authors.forename: E B
authors.initials: EB
authors.lastname: Stevens
authors.forename: R
authors.initials: R
authors.lastname: Woodward
authors.forename: I H
authors.initials: IH
authors.lastname: Ho
authors.forename: R
authors.initials: R
authors.lastname: Murrell-Lagnado
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000494:pharmacology; D004594:Electrophysiology; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008845:Microinjections; D016296:Mutagenesis / Q000502:physiology; D009865:Oocytes / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014981:Xenopus
chemical_list: D018076:DNA, Complementary; C103861:KCNJ15 protein, human; C099489:KCNJ4 protein, human; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D019204:GTP-Binding Proteins