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

Date Published: September 21, 2018

Publisher: Public Library of Science

Author(s): Michihiro Tateyama, Yoshihiro Kubo, Mark S. Shapiro.


G protein-gated inwardly rectifying K+ (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M2R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M1R with that of M2R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M1R, co-localizes with the GIRK channel and Gαi1 by Förster resonance energy transfer (FRET) analysis. When M1R was forced to stay adjacent to the channel through ligation with short linkers, M1R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M1R and the GIRK channel. The results show that co-localization is an important factor for activating the GIRK channel. In contrast, for MC9 and M2R, 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 M1R 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 M2R plays an important role in co-localization.

Partial Text

G protein-gated inwardly rectifying potassium (GIRK) channel is a key protein that regulates cellular excitability and is activated by interacting with free Gβγ released from the heterotrimeric Gαβγ complex upon the activation of G protein-coupled receptors (GPCRs) [1]. The conformational rearrangement between the GIRK channel and Gβγ has been observed upon the receptor activation on the living cell membrane by Förster or bioluminescence resonance energy transfer analyses (FRET or BRET, respectively) [2, 3] and the GIRK-Gβγ complex formation was demonstrated by structural analyses [4]. The GIRK channel is also known to bind to Gαi and Gαo in their resting and active forms [5–8], suggesting that Gβγ released from Gαi or Gαo immediately interacts with and activates the GIRK channel. In addition, several Gi/o-coupled receptors (Gi/o-Rs) have been reported to localize near Gi/o by FRET and BRET analyses [9–11]. Therefore, Gi/o-Rs, Gi/o and the GIRK channel have been suggested to form a ternary pre-signaling complex to effectively activate the GIRK channel.

We showed that M2R-i3 enables the chimeric MC9 to locate adjacent to Gαi1 and the GIRK channel, that Gq-coupled M1R activates the GIRK channel when it is in proximity to the channel, and that the distance between the receptor and GIRK channel is a key determinant in effective channel activation.




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