Both “Constitutively-active” and “Inactive” Gαi3 Mutants Interact with GIRK1/2 Heterotetramer

Abstract

Biophysical and imaging studies of the G-protein-inward-rectifying-K+-channel (GIRK), point towards the existence of multiprotein complexes of channel, G-proteins and occasionally G-Protein-coupled-receptors (GPCR). It is established that GIRK directly interacts with the Gβγ subunits, before and after receptor activation, whereas GIRK's interaction with the Gαi subunit remains unresolved. We previously reported that GIRK is closely regulated by the Gαi subunit. Biochemically, we find that the full cytoplasmic domain of the channel (G1NC) binds Gαi. This interaction is enhanced in the presence of Gβγ, with either GDP or GTPγS. Our findings demonstrate that both N- and C- termini of the channel, when associated with Gβγ, form a favorable 3D binding domain for the active and the inactive Gαi3 subunit.We assessed the interaction between GIRK and G proteins with fluorescence resonance energy transfer (FRET), using a doubly-labeled channel (DL-GIRK1), showing that the DL-GIRK channel acts as a sensitive reporter for the presence of G-proteins. DL-GIRK1 coexpressed with Gβγ displayed an increase in FRET, implying the nearing of N- and C-termini. Both phosducin and Gαi3GA (“constitutively-inactive” mutant, Gαi3G203A) caused a dramatic decrease in both currents and FRET, probably by deviating Gβγ from its activation site. Nevertheless, coexpression of Gαi3GA and Gβγ restored the channel's open conformation as reported by FRET and currents, whereas phosducin did not. An observed increase in FRET, with coexpressed Gαi3Q204L (“constitutively-active” Gαi3 mutant) and Gβγ, strongly supports our biochemical findings of constitutive interaction. We tested the effect of Gαβγ on a doubly-labeled G-protein-insensitive-inward-rectifier-K+-channel, Kir2.1 (DL-IRK1). DL-IRK1 failed to demonstrate any changes in FRET with coexpressed G-proteins.Our findings imply that GIRK acts as the nucleator of the GIRK-Gα-Gβγ complex. Both active and inactive Gαi3 remain bound to the channel, ensuring fast and specific activation and termination of the signal.