Tabak, G. | bioRxiv Science

bioRxiv2025

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

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.

physiology↗

bioRxiv2019

G protein-coupled potassium channels implicated in mouse and cellular models of GNB1 Encephalopathy

De novo mutations in GNB1, encoding the G{beta}1 subunit of G proteins, cause a neurodevelopmental disorder with global developmental delay and epilepsy. Mice carrying a pathogenic mutation, K78R, recapitulate aspects of the disorder, including developmental delay and frequent spike-wave discharges (SWD). Cultured mutant cortical neurons display aberrant bursting activity on multi-electrode arrays. Strikingly, the antiepileptic drug ethosuximide (ETX) restores normal neuronal network behavior in vitro and suppresses SWD in vivo. In contrast, while valproic acid suppresses SWD, it does not restore normal network behavior, suggesting that ETX has mechanistic specificity for the effects of aberrant G{beta}1 signaling. Consistent with this, we show that K78R is a gain-of-function of G protein-coupled potassium channel (GIRK) activation that is potently inhibited by ETX. This work suggests that altered G{beta}1 signaling causes disease in part through effects on GIRK channels, illustrates the utility of cultured neuronal networks in pharmacological screening, and establishes effective pre-clinical models for GNB1 Encephalopathy.

neuroscience↗