GNB1 encephalopathy (GNB1E) is a rare neurodevelopmental disorder caused by mutations in GNB1 gene encoding the G protein subunit Gβ1. Mechanisms linking these variants to neurological dysfunction remain unclear. We investigated the prevalent p.Ile80Thr (I80T) variant using combined clinical, cellular, and in vivo approaches. Longitudinal evaluation of a GNB1E patient revealed developmental delay, progressive peripheral spasticity, and epilepsy with Spike-Wave Activation in Sleep. Heterozygous knock-in Gnb1I80T/+ mice exhibited disease-relevant phenotypes, including impaired early development, mild adult motor and cognitive deficits and epileptiform cortical spike-and-wave discharges. Transcriptomic analysis identified 323 genes concordantly dysregulated in mouse cortex and cortical human neuronal cultures from patient-derived induced pluripotent cells. This gene set was enriched for ion-channel function, epilepsy-associated genes, and Gs/adenylyl cyclase signaling pathway. Our integrated analysis establishes the first cross-species model for GNB1E, suggests common neurological mechanisms and molecular pathways linked to GNB1E, and provides a framework for mechanistic and therapeutic studies.

BackgroundG-proteins areindispensable regulators of cellular signaling, with G-protein-gated inwardly rectifying potassium channels (GIRK) as key effectors. GNB1 encephalopathy (GNB1E) is a congenital neurological syndrome resulting from mutations in the GNB1 gene, encoding the Gβ1 subunit of G-proteins trimer (Gαβγ). GNB1E manifests as a global developmental delay, accompanied by tonus disturbances, ataxia, and epilepsy.MethodsWe utilized the Xenopus laevis oocyte heterologous expression system to investigate the impact of the L95P mutation in Gβ1 (Gβ1-L95P) on the activation of neuronal GIRK channels GIRK2 and GIRK1/2. Mutant and wild-type (WT) Gβ1 RNAs were co-injected with RNAs encoding the Gγ2 and GIRK channel subunits. The expression levels of both Gβ1 and the channel proteins, as well as the channel activity, were systematically monitored. Additionally, rigid-body docking was used to model the GIRK1/2–Gβγ complex, evaluating L95P’s effect on channel–Gβγ interaction, Gβγ stability, and Gβγ–effector affinity.Results. Gβ1-L95P exhibited reduced protein expression compared to WT. Even after RNA adjustments to restore comparable membrane localization, the mutant failed to effectively activate GIRK2 and GIRK1/2. Structural analysis revealed that L95 was not consistent in the Gβγ–effector interface. Thermodynamic calculations suggested that the mutation primarily destabilized Gβ1 and Gβ1–effector complex.ConclusionGβ1-L95P leads to both reduced protein expression and impaired function in the GIRK–Gβγ interaction system. The later effect can be attributed to the changes associated with protein misfolding.

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.

Background and PurposeThe antiepileptic drug ethosuximide (ETX) suppresses epileptiform activity in a mouse model of GNB1 syndrome, caused by mutations in Gβ1 protein, likely through the inhibition of G‐protein gated K+ (GIRK) channels. Here, we investigated the mechanism of ETX inhibition (block) of different GIRKs.Experimental ApproachWe studied ETX inhibition of GIRK channels expressed in Xenopus oocytes with or without their physiological activator, the G protein subunit dimer Gβγ. ETX binding site and mode of action were analysed using molecular dynamic (MD) simulations and kinetic modelling, and the predictions were tested by mutagenesis and functional testing.Key ResultsWe show that ETX is a subunit‐selective, allosteric blocker of GIRKs. The potency of ETX block is increased by Gβγ, in parallel with channel activation. MD simulations and mutagenesis locate the ETX binding site in GIRK2 to a region associated with phosphatidylinositol‐4,5‐bisphosphate (PIP2) regulation, and suggest that ETX acts by closing the helix bundle crossing (HBC) gate and altering channel's interaction with PIP2. The apparent affinity of ETX block is highly sensitive to changes in channel gating caused by mutations in Gβ1 or GIRK subunits.Conclusion and ImplicationsETX block of GIRKs is allosteric, subunit‐specific, and enhanced by Gβγ through an intricate network of allosteric interactions within the channel molecule. Our findings pose GIRK as a potential therapeutic target for ETX and ETX as a potent allosteric GIRK blocker and a tool for probing gating‐related conformational changes in GIRK.

G-protein coupled inwardly rectifying potassium (GIRK) channels are key players in inhibitory neurotransmission in the heart and the brain. Misregulation of GIRK is associated with a variety of neurological disorders, such as epilepsy, Alzheimer's disease, and the rare Keppen-Lubinsky syndrome. Ethosuximide (ETX) is an antiseizure drug that inhibits GIRK channels and therefore has therapeutic potential for GIRK-related disorders. Using electrophysiology experiments and μs-long molecular dynamics (MD) simulations, we identify an allosteric ETX binding site close to the binding site of the endogenous GIRK activator phosphatidylinositol 4,5-bisphosphat (PIP2).

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on beta-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage -gated calcium channels (CaV) acting through the Cav beta subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C -terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N -terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaV beta, even in the absence of CaV alpha, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C -terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaV beta, is sufficient for sympathetic upregulation of Ca2+ currents.

The adrenergic nervous system augments cardiac contraction by increasing the activity of L-type voltage-gated Ca V 1.2 channels. Dysregulation of this process is linked to severe cardiac dysfunctions. The signaling cascade involves activation of β-adrenergic receptors, elevation of cAMP levels, separation of protein kinase A (PKA) regulatory subunit (PKAR) from catalytic subunit (PKAC), and phosphorylation of the inhibitory protein Rad leading to increased Ca 2+ influx. In cardiomyocytes, the core subunit of Ca V 1.2 (α 1C ) exists in two forms: full-length (FL) or proteolytically processed (truncated), lacking the distal C-terminus (dCT). Specificity and efficiency in the cascade are believed to emanate from unique protein-protein interactions, such as anchoring PKA (via PKAR) to α 1C by A-kinase anchoring proteins (AKAPs). However, most AKAPs do not interact with the truncated α 1C , and their role in βAR regulation of cardiac Ca V 1.2 remains unclear. Here we show that PKAC, independently of PKAR or AKAPs, directly interacts with α 1C at two domains in α 1C -CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Furthermore, we find that DCRD competes with PCRD and reduces its interaction with PKAC. The physiological consequences of these complex interactions are incompletely understood; our data suggest that they may fine-tune the βAR regulation of Ca V 1.2. We propose that the newly discovered interactions take part in governing colocalization of regulatory proteins within the βAR-Ca V 1.2 multimolecular signaling complexes in cardiomyocytes.

The antiepileptic drug ethosuximide (ETX) suppresses epileptiform activity in a mouse model of GNB1 syndrome, caused by mutations in Gβ1 protein, likely through the inhibition of G-protein gated K+ (GIRK) channels. Here we show that ETX is a subunit-selective, allosteric blocker of GIRKs. The potency of ETX block is increased by the G protein subunit dimer Gβγ, the physiological activator of GIRKs. Molecular dynamics (MD) simulations and mutagenesis locate the ETX binding site in GIRK2 to a region associated with phosphatidylinositol-4,5-bisphosphate (PIP2) regulation, and suggest that ETX acts by closing the HBC gate and altering channel’s interaction with PIP2. The apparent affinity of ETX block is highly sensitive to changes in channel gating caused by mutations in Gβ1 or GIRK subunits. Our findings pose GIRK as a potential therapeutic target for ETX, and ETX as a potent allosteric GIRK blocker and a tool for probing gating-related conformational changes in GIRK. ### Competing Interest Statement The authors have declared no competing interest.

De novo mutations in GNB1, encoding the Gβ1 subunit of G proteins, cause a neurodevelopmental disorder with global developmental delay and epilepsy, GNB1 encephalopathy. Here, we show that mice carrying a pathogenic mutation, K78R, recapitulate aspects of the disorder, including developmental delay and generalized seizures. Cultured mutant cortical neurons also display aberrant bursting activity on multi-electrode arrays. Strikingly, the antiepileptic drug ethosuximide (ETX) restores normal neuronal network behavior in vitro and suppresses spike-and-wave discharges (SWD) in vivo. ETX is a known blocker of T-type voltage-gated Ca2+ channels and G protein-coupled potassium (GIRK) channels. Accordingly, we present evidence that K78R results in a gain-of-function (GoF) effect by increasing the activation of GIRK channels in cultured neurons and a heterologous model (Xenopus oocytes)—an effect we show can be potently inhibited by ETX. This work implicates a GoF mechanism for GIRK channels in epilepsy, identifies a new mechanism of action for ETX in preventing seizures, and establishes this mouse model as a pre-clinical tool for translational research with predicative value for GNB1 encephalopathy.

G-protein coupled inwardly rectifying potassium (GIRK) channels are key players in inhibitory neurotransmission in heart and brain. We conducted molecular dynamics simulations to investigate the effect of a selectivity filter (SF) mutation, G154S, on GIRK2 structure and function. We observe mutation-induced loss of selectivity, changes in ion occupancy and altered filter geometry. Unexpectedly, we reveal aberrant SF dynamics in the mutant to be correlated with motions in the binding site of the channel activator Gβγ. This coupling is corroborated by electrophysiological experiments, revealing that GIRK2wt activation by Gβγ reduces the affinity of Ba2+ block. We further present a functional characterization of the human GIRK2G154S mutant validating our computational findings. This study identifies an allosteric connection between the SF and a crucial activator binding site. This allosteric gating mechanism may also apply to other potassium channels that are modulated by accessory proteins.

The Ca V 1 and Ca V 2 families of voltage-dependent calcium channels play a crucial role in neurotransmitter release, excitation-contraction and many other cellular processes. Comprised of the membrane pore-forming α 1 , intracellular β and extracellular α 2 δ subunits, these channels have been targets for pharmacological intervention for decades. Physiological functions of Ca V channels are attenuated by either constitutively or transiently bounds proteins in the cellular environment. The RGK (Rad, Gem, Rem, and Rem2) G-protein family potently inhibits Ca V 1 and Ca V 2 function in heterologous expression systems. RGK proteins bind to Ca V β and inhibit channel localization and activity by forming a ternary complex with Ca V α 1 . Here, we evaluated the influence of RGK proteins on Ca V 2.2 channels heterologously expressed in Xenopus oocytes. Both Gem and Rad showed no nucleotide dependency on its inhibitory function on Ca V 2.2. The G-domain and C-terminus could inhibit the Ca V 2.2 channel independently when co-expressed with channel subunits. Our results demonstrated that structural determinants in Gem, crucial for channel inhibition, lie within the 222-296 amino acid region containing both the partial G-domain and C-terminus as determined from chimeric Ca V β-Gem constructs. We expanded our mapping efforts and prepared various chimeras of Drosophila melanogaster ( Dm ) RGK sequences fused to Ca V β and showed that 22 residues in RGK2t and RGK3L C-terminal imparted complete Ca V 2.2 inhibition. Point mutations in the Dm RGK C-terminus, conserved in mammalian RGK proteins, abrogated the Ca V 2.2 inhibition to a significant extent, pointing to a hot region in the extreme C-terminus for inhibition of Ca V channels. Since RGK homologs are now recognized as physiological modulators in β-adrenergic regulation of Ca V channels, the relevance of this curious G-protein family deserves close examination.

G protein-sensitive inwardly rectifying potassium (GIRK) channels are important pharmaceutical targets for neuronal, cardiac, and endocrine diseases. Although a number of GIRK channel modulators have been discovered in recent years, most lack selectivity. GIRK channels function as either homomeric (i.e., GIRK2 and GIRK4) or heteromeric (e.g., GIRK1/2, GIRK1/ 4, and GIRK2/3) tetramers. Activators, such as ML297, ivermectin, and GAT1508, have been shown to activate heteromeric GIRK1/2 channels better than GIRK1/4 channels with varying degrees of selectivity but not homomeric GIRK2 and GIRK4 channels. In addition, VU0529331 was discovered as the first homomeric GIRK channel activator, but it shows weak selectivity for GIRK2 over GIRK4 (or G4) homomeric channels. Here, we report the first highly selective small-molecule activator targeting GIRK4 homomeric channels, 3hi2one-G4 (3-[2(3,4-dimethoxyphenyl)-2-oxoethyl]-3-hydroxy-1-(1-naphthylm ethyl)-1,3-dihydro-2H-indol-2-one). We show that 3hi2one-G4 does not activate GIRK2, GIRK1/2, or GIRK1/4 channels. Using molecular modeling, mutagenesis, and electrophysiology, we analyzed the binding site of 3hi2one-G4 formed by the trans membrane 1, transmembrane 2, and slide helix regions of the GIRK4 channel, near the phosphatidylinositol-4,5bisphosphate binding site, and show that it causes channel activation by strengthening channel-phosphatidylinositol-4,5-bisphosphate interactions. We also identify slide helix residue L77 in GIRK4, corresponding to residue I82 in GIRK2, as a major determinant of isoform-specific selectivity. We propose that 3hi2one-G4 could serve as a useful pharmaceutical probe in studying GIRK4 channel function and may also be pursued in drug optimization studies to tackle GIRK4-related diseases such as primary aldosteronism and late-onset obesity.

Mutations in the GNB1 gene, encoding the Gβ1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gβ1 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 Gi/o, or the Gβγ regulation of the neuronal voltage-gated Ca2+ channel CaV2.2. However, the mutations profoundly affected the Gβγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gβ1 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.

G-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 Ca2+ 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 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 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-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. Significance Statement Mutations 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 gain-of-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.

Significance The strengthening of heart contraction by epinephrine (adrenaline) and norepinephrine starts with the activation of β-adrenergic receptors and culminates in a protein kinase A–mediated increase in Ca 2+ influx through the voltage-gated Ca 2+ channel, Ca V 1.2, into cardiomyocytes. Many crucial molecular details of this vital physiological regulation remained enigmatic for decades, not the least owing to the difficulty of reconstituting the regulation in model cells. Capitalizing on the recent discovery of the central role of the Ca V 1.2-associated protein, Rad, we present a report of full reconstitution of the β-AR–Ca V 1.2 cascade in a model system, the Xenopus oocyte, and investigate crucial aspects of regulation such as the roles of auxiliary subunits and diverse forms of the channel protein.

The G protein-activated Inwardly Rectifying K+-channel (GIRK) modulates heart rate and neuronal excitability. Following G-Protein Coupled Receptor (GPCR)-mediated activation of heterotrimeric G proteins (Gαβγ), opening of the channel is obtained by direct binding of Gβγ subunits. Interestingly, GIRKs are solely activated by Gβγ subunits released from Gαi/o-coupled GPCRs, despite the fact that all receptor types, for instance Gαq-coupled, are also able to provide Gβγ subunits. It is proposed that this specificity and fast kinetics of activation stem from pre-coupling (or pre-assembly) of proteins within this signaling cascade. However, many studies, including our own, point towards a diffusion-limited mechanism, namely collision coupling. Here, we set out to address this long-standing question by combining electrophysiology, imaging, and mathematical modeling. Muscarinic-2 receptors (M2R) and neuronal GIRK1/2 channels were coexpressed in Xenopus laevis oocytes, where we monitored protein surface expression, current amplitude, and activation kinetics. Densities of expressed M2R were assessed using a fluorescently labeled GIRK channel as a molecular ruler. We then incorporated our results, along with available kinetic data reported for the G-protein cycle and for GIRK1/2 activation, to generate a comprehensive mathematical model for the M2R-G-protein-GIRK1/2 signaling cascade. We find that, without assuming any irreversible interactions, our collision coupling kinetic model faithfully reproduces the rate of channel activation, the changes in agonist-evoked currents and the acceleration of channel activation by increased receptor densities.