Two Distinct Aspects of Coupling between Gαi Protein and G Protein-activated K+ Channel (GIRK) Revealed by Fluorescently Labeled Gαi3 Protein Subunits

G protein-activated K+ channels (Kir3 or GIRK) are activated by direct interaction with G beta gamma. G alpha 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-G alpha-G beta gamma signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-G beta gamma interactions but yielded controversial results regarding the GIRK-G alpha(i/o) interaction. We investigated the mechanisms of regulation of GIRK by G alpha(i/o) using wild-type G alpha(i3) (G alpha i3WT) and G alpha(i3) labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). G alpha(i3)xFP proteins bound the cytosolic domain of GIRK1 and interacted with G beta gamma in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated G alpha(i3)xFP (G alpha i3NT) closely mimicked all aspects of G alpha i3WT regulation except for a weaker regulation of I-basal. G alpha(i3) labeled with YFP within the G alpha helical domain preserved regulation of I-basal but failed to restore fast I-evoked. Titrated expression of G alpha i3NT and G alpha(i3) WT confirmed that regulation of I-basal and of the kinetics of I-evoked of GIRK1/2 are independent functions of G alpha(i). FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and G beta gamma than between GIRK1 and G alpha(i3). Thus, G alpha(i/o)beta gamma heterotrimer may be attached to GIRK primarily via G beta gamma within the signaling complex. Our findings support the notion that G alpha(i/o) actively regulates GIRK. Although regulation of I-basal is a function of G alpha(GDP)(i), our new findings indicate that regulation of kinetics of I-evoked is mediated by G alpha(GTP)(i).