Mapping the G  -binding Sites in GIRK1 and GIRK2 Subunits of the G Protein-activated K+ Channel (original) (raw)

G protein-activated K ؉ channels (Kir3 or GIRK) are activated by direct binding of G␤␥. The binding sites of G␤␥ in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of G␤␥ has not been studied. We verified and extended the map of G␤␥-binding sites in GIRK1 by using two approaches: direct binding of G␤␥ to fragments of GIRK subunits (pull down), and competition of these fragments with the G␣ i1 subunit for binding to G␤␥. We also mapped the G␤␥-binding sites in GIRK2. In both subunits, the N terminus binds G␤␥. In the C terminus, the G␤␥-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized G␤␥-interacting segments in the first half of the C terminus. The main C-terminal G␤␥-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 G␤␥ 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 G␤␥ to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of G␤␥-induced changes in channel gating rather than G␤␥ binding.