Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels

G_i/o protein-coupled receptors (GPCRs) inhibit cardiac and neuronal excitability via G protein-activated K⁺ channels (GIRK), assembled by combinations of GIRK1 - GIRK4 subunits. GIRKs are activated by direct binding of the Gβγ dimer of inhibitory G_i/o proteins. However, key aspects of this textbook signaling pathway remain debated. Recent studies suggested no G_i/o-GIRK pre-coupling and low (>250 µM) Gβγ-GIRK interaction affinity, contradicting earlier sub-µM estimates and implying low signaling efficiency. We show that Gγ prenylation, which mediates Gβγ membrane attachment required for GIRK activation, also contributes to the Gβγ-GIRK interaction, explaining the poor affinity obtained with non-prenylated Gβγ. Using quantitative protein titration and electrophysiology in live Xenopus oocytes, Gβγ affinity for homotetrameric GIRK2 ranges from 4-30 µM. Heterotetrameric GIRK1/2 shows a higher Gβγ apparent affinity due to the Gβγ-docking site (anchor) in GIRK1, which enriches Gβγ at the channel. Biochemical approaches and molecular dynamic simulations reveal that the Gβγ anchor is formed by interacting N-terminal and distal C-terminal domains of the GIRK1 subunits, distinct from the Gβγ-binding “activation” site(s) underlying channel opening. Thus, the affinity of Gβγ-GIRK interaction is within the expected physiological range, while dynamic pre-coupling of Gβγ to GIRK1-containing channels through high-affinity interactions further enhances the GPCR-G_i/o-GIRK signaling efficiency.