Gα_i and Gβγ jointly regulate the conformations of a Gβγ effector, the neuronal G protein-activated K⁺ channel (GIRK)

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical Gβγ effector G protein-activated K⁺ channel (GIRK; Kir3) physically interacts with Gβγ but also with Gα_i/o. Whether and how Gα_i/o subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, Gα_i3 and Gβγ. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that Gβγ and Gα_i3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that Gβγ greatly enhances the binding of GIRK1 subunit to Gα_i3^GDP and, unexpectedly, to Gα_i3^GTP. i-FRET showed that both Gα_i3 and Gβγ induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a "constitutively active" Gα_i3 mutant andGβγ together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either Gα_i3 or Gβγ. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-Gα-Gβγ signaling complex and mediates allosteric interactions between Gα_i^GTP and Gβγ. Our findings imply that Gα_i/o and the Gα_iβγ heterotrimer can regulate a Gβγ effector both before and after activation by neurotransmitters.