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<p>G<sub>i/o</sub> protein-coupled receptors (GPCRs) inhibit cardiac and neuronal excitability via G protein-activated K<sup>+</sup> channels (GIRK), assembled by combinations of GIRK1 - GIRK4 subunits. GIRKs are activated by direct binding of the Gβγ dimer of inhibitory G<sub>i/o</sub> proteins. However, key aspects of this textbook signaling pathway remain debated. Recent studies suggested no G<sub>i/o</sub>-GIRK pre-coupling and low (&gt;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 <i>Xenopus</i> 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<sub>i/o</sub>-GIRK signaling efficiency.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月24日

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

摘要

Gi/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 Gi/o proteins. However, key aspects of this textbook signaling pathway remain debated. Recent studies suggested no Gi/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-Gi/o-GIRK signaling efficiency.