Pertussis-toxin-sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex.
Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (Galphai/o) G proteins. Although the Gbetagamma dimer is known to activate GIRK channels, the role of the Galphai/o subunit remains unclear. Here, we established that Galphao subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for Galphao binding. Further studies suggested that the Galphai/obetagamma heterotrimer binds to the GIRK2 C-terminal domain via Galpha and not Gbetagamma. Galphai/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and Gbetagamma-activated currents. Finally, PTX-insensitive Galphaq or Galphas subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive Galphai/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific Galphai/o signaling pathways.
Clancy
Sinead M
SM
Peptide Biology Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Fowler
Catherine E
CE
Finley
Melissa
M
Suen
Ka Fai
KF
Arrabit
Christine
C
Berton
Frédérique
F
Kosaza
Tohru
T
Casey
Patrick J
PJ
Slesinger
Paul A
PA
D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044385:GTP-Binding Protein alpha Subunits / Q000187:drug effects / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000187:drug effects / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D009865:Oocytes; D037342:Pertussis Toxin / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014981:Xenopus
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D024661:Potassium Channels, Inwardly Rectifying; D043562:Receptors, G-Protein-Coupled; D000431:Ethanol; D037342:Pertussis Toxin; D020962:Heterotrimeric GTP-Binding Proteins