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

Handklo-Jamal, Reem; Raifman, Tal Keren; Shalomov, Boris; Hofer, Patrick; Kahanovitch, Uri; Friesacher, Theres; Tabak, Galit; Tsemakhovich, Vladimir; Reddy, Haritha P.; Chomsky-Hecht, Orna; Tripathy, Debi Ranjan; Zuhlke, Kerstin; Dessauer, Carmen W.; Klussmann, Enno; Haitin, Yoni; Hirsch, Joel A.; Stary-Weinzinger, Anna; Yakubovich, Daniel; Dascal, Nathan

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

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Item Type:	Article
Title:	Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels
Creators Name:	Handklo-Jamal, Reem, Raifman, Tal Keren, Shalomov, Boris, Hofer, Patrick, Kahanovitch, Uri, Friesacher, Theres, Tabak, Galit, Tsemakhovich, Vladimir, Reddy, Haritha P., Chomsky-Hecht, Orna, Tripathy, Debi Ranjan, Zuhlke, Kerstin, Dessauer, Carmen W., Klussmann, Enno, Haitin, Yoni, Hirsch, Joel A., Stary-Weinzinger, Anna, Yakubovich, Daniel and Dascal, Nathan
Abstract:	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.
Source:	Nature Communications
ISSN:	2041-1723
Publisher:	Nature Publishing Group
Date:	24 November 2025
Official Publication:	https://doi.org/10.1038/s41467-025-66730-8
PubMed:	View item in PubMed
Related to:	URL URL Type https://doi.org/10.5281/zenodo.15075372 External Dataset https://www.wwpdb.org/pdb?id=pdb_00005elu External Dataset https://www.wwpdb.org/pdb?id=pdb_00001gp2 External Dataset

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