G protein-coupled inward rectifier K+ channels (GIRK channels) are activated directly by the G protein Py subunit, The crystal structure of the G protein py subunits reveals that the beta subunit consists of an N-terminal a helix followed by a symmetrical seven-bladed propeller structure. Each blade is made up of four antiparallel P strands. The top surface of the propeller structure interacts with the G alpha subunit, The outer surface of the py torus is largely made from outer beta strands of the propeller. We analyzed the interaction between the beta subunit and brain GIRK channels by mutating the outer surface of the py torus, Mutants of the outer surface of the beta, subunit were generated by replacing the sequences at the outer p strands of each blade with corresponding sequences of the yeast P subunit, STE4, The mutant beta (1)gamma (2) subunits were expressed in and purified from Sf9 cells. They were applied to inside-out patches of cultured locus coeruleus neurons, The wild type Ply, induced robust GIRK channel activity with an EC50 of about 4 nM, Among the eight outer surface mutants tested, blade 1 and blade 2 mutants (D1 and CD2) were far less active than the wild type in stimulating GIRK channels. However, the ability of I)1 and CD2 to regulate type I and type II adenylyl cyclases was not very different from that of the wild type beta (1)gamma (2), As to the activities to stimulate phospholipase C beta (2) D1 was more potent and CD2 was less potent than the wild type beta (1)gamma (2),. Additionally we tested four beta (1) mutants in which mutated residues are located in the top G alpha/beta interacting surface, Among them, mutant W332A showed far less ability than the wild type to activate GIRK channels. These results suggest that the outer surface of blade 1 and blade 2 of the beta subunit might specifically interact with GIRK and that the beta subunit interacts with GIRK both over the outer surface and over the top G alpha interacting surface.
