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| Item Type: | Preprint |
|---|---|
| Title: | Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels |
| Creators Name: | Handklo-Jamal, Reem, Keren-Raifman, Tal, Shalomov, Boris, Hofer, Patrick, Kahanovitch, Uri, Friesacher, Theres, Tabak, Galit, Tsemakhovich, Vladimir, Reddy, Haritha P., Chomsky-Hecht, Orna, Ranjan Tripathy, Debi, Zuhlke, Kerstin, Dessauer, Carmen W., Klussmann, Enno, Haitin, Yoni, Hirsch, Joel, Stary-Weinzinger, Anna, Yakubovich, Daniel and Dascal, Nathan |
| Abstract: | G(i/o) protein-coupled receptors (GPCRs) inhibit cardiac and neuronal excitability via G proteinactivated 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 precoupling 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 ranged from 4-30 µM. Heterotetrameric GIRK1/2 showed a higher Gβγ apparent affinity due to unique Gβγdocking site (anchor) in GIRK1, which enriches Gβγ at the channel. Biochemical approaches and molecular dynamic simulations revealed 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. |
| Keywords: | Animals, Frogs |
| Source: | bioRxiv |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Article Number: | 2025.10.15.682510 |
| Date: | 15 October 2025 |
| Official Publication: | https://doi.org/10.1101/2025.10.15.682510 |
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