Divergent regulation of GIRK1 and GIRK2 subunits of the neuronal G protein gated K⁺ channel by Gα_i^GDP and Gβγ

G protein activated K⁺ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of G_i/o proteins via G protein-coupled receptors (GPCRs). Although Gα i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gα_i3 decreases the basal activity (I_basal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gα_i3^GDP and involves the formation of Gα_i3βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ 'away' from the channel. The regulation of basal and Gβγ-evoked current was produced by the 'constitutively inactive'mutant of Gα_i3, Gα_i3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gα_i3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gα_i3 G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gα_i3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gα_i3G203A did not affect either I_basal or Gβγ-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large I_basal and was poorly activated by Gβγ. Expression of Gα_i3 GDP reduced I_basal and restored the ability of Gβγ to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both Gα_i3 ^GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gα_i3 ^GDP.