De novo mutations in GNB1, encoding the Gβ1 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 Gβ1 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 Gβ1 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.

Summary Mutations in the GNB1 gene, encoding the Gβ 1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gβ 1 mutations (K78R, I80N and I80T) using computational and functional approaches. In heterologous expression models, these mutations did not alter the coupling between G protein-coupled receptors to G i/o , or the Gβγ regulation of the neuronal voltage-gated Ca 2+ channel Ca V 2.2. However, the mutations profoundly affected the Gβγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gβ 1 protein expression levels, Gβγ binding to cytosolic segments of GIRK subunits, and in Gβγ function, and included gain-of-function for K78R or loss-of-function for I80T/N, which were GIRK subunit-specific. Our findings offer new insights into subunit-dependent gating of GIRKs by Gβγ, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment.

ClassificationBiological Sciences: Physiology. AbstractG-protein gated, inwardly rectifying potassium channels (GIRK) mediate inhibitory transmission in brain, heart, and adrenal cortex. GIRK4 (KCNJ5) subunits are abundant in the heart and adrenal cortex. Multiple mutations of KCNJ5 cause primary aldosteronism (PA). According to a leading concept, mutations in the pore region of GIRK4 cause loss of K + selectivity; the ensuing Na + influx depolarizes zona glomerulosa cells and activates voltage gated Ca 2+ channels, inducing hypersecretion of aldosterone. The concept of selectivity loss has been extended to mutations in cytosolic domains of GIRK4 channels, remote from the pore region. We expressed GIRK4R52H, GIRK4E246K, and GIRK4G247R mutants in Xenopus oocytes and human adrenocortical carcinoma cell line (HAC15). Whole-cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K (but not GIRK1/4G247R) channels were greatly reduced compared to GIRK1/4WT. Nevertheless, all heterotetrameric mutants retained full K + selectivity and inward rectification. When expressed as homotetramers, only GIRK4WT, but none of the mutants, produced wholecell currents. Confocal imaging, single channel and Förster Resonance Energy Transfer (FRET) analyses showed: 1) reduction of membrane abundance of all mutated channels, especially as homotetramers, 2) impaired interaction with Gβγ subunits, and 3) reduced open probability of GIRK1/4R52H. VU0529331, a GIRK4 opener, activated homotetrameric GIRK4G247R channels, but not GIRK4R52H and GIRK4E246K. Our results suggest impaired gating (GIRK4R52H) and expression in plasma membrane (all mutants). We suggest that, contrary to the previously proposed mechanism, R52H and E246K mutants are loss-offunction rather than gain-of-function/selectivity-loss mutants. Hence, GIRK4 openers may be a potential course of treatment for patients with cytosolic N-and C-terminal mutations. Significance StatementMutations in KCNJ5 gene, which encodes for the GIRK4 subunit of G-protein inwardly rectifying K+ channels, are the main cause of primary aldosteronism, a major contributor to secondary hypertension. We report that three mutations in the cytosolic domain of GIRK4 cause loss-of-function, contrary to the prevailing concept that these mutations cause loss of selectivity and subsequent depolarization, i.e. essentially gainof-function. Our findings correct the existing misconception regarding the biophysical mechanism that impairs the channel function, and may provide indications for future personalized treatment of the disease.

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G protein‐gated, inwardly rectifying potassium channels (GIRK) mediate inhibitory transmission in brain and heart, and are present in the adrenal cortex. GIRK4 (KCNJ5) subunits are abundant in the heart and adrenal cortex. Multiple mutations of KCNJ5 cause primary aldosteronism (PA). Mutations in the pore region of GIRK4 cause loss of K+ selectivity, Na+ influx and depolarization of zona glomerulosa cells followed by hypersecretion of aldosterone. The concept of selectivity loss has been extended to mutations in cytosolic domains of GIRK4 channels, remote from the pore. We expressed aldosteronism‐linked GIRK4R52H, GIRK4E246K and GIRK4G247R mutants in Xenopus oocytes. Whole‐cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K channels were greatly reduced compared with GIRK1/4WT. Nevertheless, all heterotetrameric mutants retained full K+ selectivity and inward rectification. When expressed as homotetramers, only GIRK4WT, but none of the mutants, produced whole‐cell currents. Confocal imaging, single‐channel and Förster Resonance Energy Transfer (FRET) analyses showed: (1) reduction of membrane abundance of all mutated channels, especially as homotetramers, (2) impaired interaction with Gβγ subunits, and (3) reduced open probability of GIRK1/4R52H. VU0529331, a GIRK4 opener, activated homotetrameric GIRK4G247R channels, but not GIRK4R52H or GIRK4E246K. In the human adrenocortical carcinoma cell line (HAC15), VU0529331 and overexpression of heterotetrameric GIRK1/4WT, but not overexpression of GIRK1/4 mutants, reduced aldosterone secretion. Our results suggest that, contrary to pore mutants of GIRK4, non‐pore mutants R52H and E246K mutants are loss‐of‐function rather than gain‐of‐function/selectivity‐loss mutants. Hence, GIRK4 openers may be a potential course of treatment for patients with cytosolic N‐ and C‐terminal mutations. Key points Mutations in GIRK4 (KCNJ5) G protein‐gated channels cause primary aldosteronism, a major cause of secondary hypertension. The primary mechanism is believed to be loss of K+ selectivity. R52H and E246K, aldosteronism‐causing mutations in cytosolic N‐ and C‐ termini of GIRK4, were reported to cause loss of K+ selectivity. We show that R52H, E246K and G247R mutations render homotetrameric GIRK channels non‐functional. In heterotetrameric context with GIRK1, these mutations impair membrane expression, interaction with Gβγ and open probability, but do not alter K+ selectivity or inward rectification. In the human aldosterone‐secreting cell line, a GIRK4 opener and overexpression of heterotetrameric GIRK1/4WT, but not overexpression of GIRK1/4 mutants, reduced aldosterone secretion. Aldosteronism‐causing mutations in the cytosolic domain of GIRK4 are loss‐of‐function mutations rather than gain‐of‐function, selectivity‐loss mutations. Deciphering of exact biophysical mechanism that impairs the channel is crucial for setting the course of treatment.