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

Abstract

G protein-gated inwardly rectifying K ⁺ (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M ₂R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M ₁R with that of M ₂R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M ₁R, co-localizes with the GIRK channel and Gα _i1 by Förster resonance energy transfer (FRET) analysis. When M ₁R was forced to stay adjacent to the channel through ligation with short linkers, M ₁R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M ₁R 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 ₂R, 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 ₁R 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 ₂R plays an important role in co-localization.