Gαi 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 G beta gamma effector G protein-activated K+ channel (GIRK; Kir3) physically interacts with G beta gamma but also with G alpha(i/o). Whether and how G alpha(i/o) subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, G alpha(i3) and G beta gamma. We used in vitro protein interaction assays and in vivo intramolecular Forster 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 G beta gamma and G alpha i3 distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G beta gamma greatly enhances the binding of GIRK1 subunit to G i3 GDP and, unexpectedly, to G alpha(GTP)(i3), i-FRET showed that both G alpha(i3) and G beta gamma induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a constitutively active G alpha(i3) mutant and G beta gamma together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G alpha(i3) or G beta gamma. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G alpha-G beta gamma signaling complex and mediates allosteric interactions between G alpha(GTP)(i3) and G beta gamma. Our findings imply that G alpha(i/o) and the G alpha(i)beta gamma heterotrimer can regulate a G beta gamma effector both before and after activation by neurotransmitters.