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

摘要

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 subunit of G(i/o) proteins. Heterologously expressed GIRK1/2 exhibit high, G-dependent basal currents (I-basal) and a modest activation by GPCR or coexpressed G. Inversely, the GIRK2 homotetramers exhibit low I-basal and strong activation by G. The high I-basal 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 (a phenomenon we term G recruitment') but not of coexpressed G(i3). All GIRK1-containing channels, but not GIRK2 homomers, recruited G to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and G, but not G(i3). Nevertheless, the truncation of G1-dCT does not impair activation by G. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes G recruitment and decreases I-basal. Thus, we conclude that G1-dCT carries an essential role in G recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a G anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by G beta gamma.