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

• Published in: The Journal of physiology Vol. 592; no. 24; pp. 5373 - 5390
• Main Authors: Kahanovitch, Uri, Tsemakhovich, Vladimir, Berlin, Shai, Rubinstein, Moran, Styr, Boaz, Castel, Ruth, Peleg, Sagit, Tabak, Galit, Dessauer, Carmen W, Ivanina, Tatiana, Dascal, Nathan
• Format: Journal Article
• Language: English
• Published: 15.12.2014
• Subjects: Xenopus laevis
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2014.283218

The G-protein coupled inwardly rectifying potassium (GIRK) channel is an important mediator of neurotransmission via G beta gamma subunit of the heterotrimeric Gi/o protein released by G-protein coupled receptor (GPCR) activation.Channels containing the GIRK1 subunit exhibit high basal currents, whereas channels that are formed by the GIRK2 subunit have very low basal currents.GIRK1-containing channels, but not channels consisting of GIRK2 only, recruit G beta gamma to the plasma membrane. The G alpha subunit of the G protein is not recruited by either GIRK1/2 or GIRK2.The unique distal C terminus of GIRK1 (G1-dCT) endows the channel with strong interaction with G beta gamma , and deletion of G1-dCT abolishes the G beta gamma recruitment and reduces the basal currents.These findings suggest that the basal activity of GIRK channels depends on channel-induced recruitment of G beta gamma . The unique C terminus of GIRK1 subunit plays an important role in G beta gamma recruitment. 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 G beta gamma subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, G beta gamma -dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed G beta gamma . Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by G beta gamma . 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 G beta gamma (a phenomenon we term 'G beta gamma recruitment') but not of coexpressed G alpha i3. All GIRK1-containing channels, but not GIRK2 homomers, recruited G beta gamma to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and G beta gamma , but not G alpha i3. Nevertheless, the truncation of G1-dCT does not impair activation by G beta gamma . In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes G beta gamma recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in G beta gamma recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a G beta gamma anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by G beta gamma .