Mapping the Gbetagamma-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel.
G protein-activated K+ channels (Kir3 or GIRK) are activated by direct binding of Gbetagamma. The binding sites of Gbetagamma in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of Gbetagamma has not been studied. We verified and extended the map of Gbetagamma-binding sites in GIRK1 by using two approaches: direct binding of Gbetagamma to fragments of GIRK subunits (pull down), and competition of these fragments with the Galphai1 subunit for binding to Gbetagamma. We also mapped the Gbetagamma-binding sites in GIRK2. In both subunits, the N terminus binds Gbetagamma. In the C terminus, the Gbetagamma-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized Gbetagamma-interacting segments in the first half of the C terminus. The main C-terminal Gbetagamma-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for Gbetagamma regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K+ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of Gbetagamma to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of Gbetagamma-induced changes in channel gating rather than Gbetagamma binding.
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.
Tatiana
T
Ivanina
Ida
I
Rishal
Dalia
D
Varon
Carmen
C
Mullner
Bibiane
B
Frohnwieser-Steinecke
Wolfgang
W
Schreibmayer
Carmen W
CW
Dessauer
Nathan
N
Dascal
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D004553:Electric Conductivity; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D015640:Ion Channel Gating; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D014981:Xenopus
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; C497066:KCNJ3 protein, human; D010446:Peptide Fragments; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D020962:Heterotrimeric GTP-Binding Proteins