Recruitment of Gßγ controls the basal activity of G-protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1.

The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1-4), activated by direct binding of the Gßγ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gßγ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gßγ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gßγ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gßγ (a phenomenon we term 'Gßγ recruitment') but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gßγ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gßγ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gßγ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gßγ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gßγ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gßγ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gßγ.

Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits.

G protein-activated K(+) channels (Kir3 or GIRK) are activated by direct interaction with Gßγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I(basal)) and kinetics of the response elicited by activation by G protein-coupled receptors (I(evoked)). These regulations are believed to occur within a GIRK-Gα-Gßγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gßγ interactions but yielded controversial results regarding the GIRK-Gα(i/o) interaction. We investigated the mechanisms of regulation of GIRK by Gα(i/o) using wild-type Gα(i3) (Gα(i3)WT) and Gα(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gßγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα(i3)xFP (Gα(i3)NT) closely mimicked all aspects of Gα(i3)WT regulation except for a weaker regulation of I(basal). Gα(i3) labeled with YFP within the Gα helical domain preserved regulation of I(basal) but failed to restore fast I(evoked). Titrated expression of Gα(i3)NT and Gα(i3)WT confirmed that regulation of I(basal) and of the kinetics of I(evoked) of GIRK1/2 are independent functions of Gα(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gßγ than between GIRK1 and Gα(i3). Thus, Gα(i/o)ßγ heterotrimer may be attached to GIRK primarily via Gßγ within the signaling complex. Our findings support the notion that Gα(i/o) actively regulates GIRK. Although regulation of I(basal) is a function of Gα(i)(GDP), our new findings indicate that regulation of kinetics of I(evoked) is mediated by Gα(i)(GTP).

G alpha(i) and G betagamma jointly regulate the conformations of a G betagamma 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 betagamma effector G protein-activated K(+) channel (GIRK; Kir3) physically interacts with G betagamma 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 betagamma. 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 G betagamma and G alpha(i3) distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G betagamma greatly enhances the binding of GIRK1 subunit to G alpha(i3)(GDP) and, unexpectedly, to G alpha(i3)(GTP). i-FRET showed that both G alpha(i3) and G betagamma 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 betagamma together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G alpha(i3) or G betagamma. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G alpha-G betagamma signaling complex and mediates allosteric interactions between G alpha(i)(GTP) and G betagamma. Our findings imply that G alpha(i/o) and the G alpha(i) betagamma heterotrimer can regulate a G betagamma effector both before and after activation by neurotransmitters.