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bioRxiv · 10.1101/2025.10.15.682510

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

Handklo-Jamal, R.·Keren-Raifman, T.·Shalomov, B.·Hofer, P.·Kahanovitch, U.·Friesacher, T.·Tabak, G.·Tsemakhovich, V.·Reddy, H. P.·Chomsky-Hecht, O.·Ranjan Tripathy, D.·Zuhlke, K.·Dessauer, C. W.·Klussmann, E.·Haitin, Y.·Hirsch, J.·Weinzinger, A. S.·Yakubovich, D.·Dascal, N.
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Abstract

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 G{beta}{gamma} 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 {micro}M) G{beta}{gamma}-GIRK interaction affinity, contradicting earlier sub-{micro}M estimates and implying low signaling efficiency. We show that G{gamma} prenylation, which mediates G{beta}{gamma} membrane attachment required for GIRK activation, also contributes to the G{beta}{gamma}-GIRK interaction, explaining the poor affinity obtained with non-prenylated G{beta}{gamma}. Using quantitative protein titration and electrophysiology in live Xenopus oocytes, G{beta}{gamma} affinity for homotetrameric GIRK2 ranged from 4-30 {micro}M. Heterotetrameric GIRK1/2 showed a higher G{beta}{gamma} apparent affinity due to unique G{beta}{gamma}-docking site (anchor) in GIRK1, which enriches G{beta}{gamma} at the channel. Biochemical approaches and molecular dynamic simulations revealed that the G{beta}{gamma} anchor is formed by interacting N-terminal and distal C-terminal domains of the GIRK1 subunits, distinct from the G{beta}{gamma}-binding "activation" site(s) underlying channel opening. Thus, the affinity of G{beta}{gamma}-GIRK interaction is within the expected physiological range, while dynamic pre-coupling of G{beta}{gamma} to GIRK1-containing channels through high-affinity interactions further enhances the GPCR-Gi/o-GIRK signaling efficiency.

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Handklo-Jamal, R., Keren-Raifman, T., Shalomov, B., Hofer, P., Kahanovitch, U., Friesacher, T., Tabak, G., Tsemakhovich, V., Reddy, H. P., Chomsky-Hecht, O., Ranjan Tripathy, D., Zuhlke, K., Dessauer, C. W., Klussmann, E., Haitin, Y., Hirsch, J., Weinzinger, A. S., Yakubovich, D., Dascal, N.. 2025-10-15. Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels. https://doi.org/10.1101/2025.10.15.682510

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