Gi/o-coupled muscarinic receptors co-localize with GIRK channel for efficient channel activation

G protein-gated inwardly rectifying K(+) (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M(2)R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M(1)R with that of M(2)R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M(1)R, co-localizes with the GIRK channel and Gα(i1) by Förster resonance energy transfer (FRET) analysis. When M(1)R was forced to stay adjacent to the channel through ligation with short linkers, M(1)R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M(1)R and the GIRK channel. The results show that co-localization is an important factor for activating the GIRK channel. In contrast, for MC9 and M(2)R, the GIRK channel was activated even when they were connected by long linkers, suggesting the formation of a molecular complex even in the absence of a linker. We also observed that replacement of 13 amino acid residues at the N-terminal end of i3 of MC9 with those of M(1)R impaired the co-localization with the GIRK channel as well as channel activation. These results show that localization of the receptor near the GIRK channel is a key factor in efficiently activating the channel and that the N-terminal end of i3 of M(2)R plays an important role in co-localization.