{"doi":"10.1101/2025.10.15.682510","title":"Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels","abstract":"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 ranged from 4-30 µM. Heterotetrameric GIRK1/2 showed a higher Gβγ apparent affinity due to unique Gβγ-docking 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":578131,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9512,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":733087,"name":"Tal Keren‐Raifman","orcid":null,"position":1,"is_corresponding":false},{"id":732301,"name":"Boris Shalomov","orcid":"0000-0002-1322-4906","position":2,"is_corresponding":false},{"id":1488183,"name":"Patrick Hofer","orcid":null,"position":3,"is_corresponding":false},{"id":432145,"name":"Uri Kahanovitch","orcid":"0000-0002-6203-7103","position":4,"is_corresponding":false},{"id":1487809,"name":"Theres Friesacher","orcid":"0000-0001-5632-3472","position":5,"is_corresponding":false},{"id":732299,"name":"Galit Tabak","orcid":"0000-0003-0414-0847","position":6,"is_corresponding":false},{"id":732300,"name":"Vladimir Tsemakhovich","orcid":"0000-0002-4969-6497","position":7,"is_corresponding":false},{"id":732297,"name":"Haritha P. 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