Epilepsy in a mouse model of GNB1 encephalopathy arises from altered potassium (GIRK) channel signaling and is alleviated by a GIRK inhibitor.

De novo mutations in GNB1, encoding the Gß1 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.

A selectivity filter mutation provides insights into gating regulation of a K+ channel.

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 Gßγ. This coupling is corroborated by electrophysiological experiments, revealing that GIRK2wt activation by Gßγ 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.

A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5).

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 Gßγ 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 Gßγ 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.

Encephalopathy-causing mutations in Gß1 (GNB1) alter regulation of neuronal GIRK channels.

Mutations in the GNB1 gene, encoding the Gß1 subunit of heterotrimeric G proteins, cause GNB1 Encephalopathy. Patients experience seizures, pointing to abnormal activity of ion channels or neurotransmitter receptors. We studied three Gß1 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 Gßγ regulation of the neuronal voltage-gated Ca2+ channel CaV2.2. However, the mutations profoundly affected the Gßγ regulation of the G protein-gated inwardly rectifying potassium channels (GIRK, or Kir3). Changes were observed in Gß1 protein expression levels, Gßγ binding to cytosolic segments of GIRK subunits, and in Gßγ 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 Gßγ, and indicate diverse etiology of GNB1 Encephalopathy cases, bearing a potential for personalized treatment.

The Neuropeptide Orexin-A Inhibits the GABAA Receptor by PKC and Ca2+/CaMKII-Dependent Phosphorylation of Its ß1 Subunit.

Orexin-A and orexin-B (Ox-A, Ox-B) are neuropeptides produced by a small number of neurons that originate in the hypothalamus and project widely in the brain. Only discovered in 1998, the orexins are already known to regulate several behaviours. Most prominently, they help to stabilise the waking state, a role with demonstrated significance in the clinical management of narcolepsy and insomnia. Orexins bind to G-protein-coupled receptors (predominantly postsynaptic) of two subtypes, OX1R and OX2R. The primary effect of Ox-OXR binding is a direct depolarising influence mediated by cell membrane cation channels, but a wide variety of secondary effects, both pre- and postsynaptic, are also emerging. Given that inhibitory GABAergic neurons also influence orexin-regulated behaviours, crosstalk between the two systems is expected, but at the cellular level, little is known and possible mechanisms remain unidentified. Here, we have used an expression system approach to examine the feasibility, and nature, of possible postsynaptic crosstalk between Ox-A and the GABAA receptor (GABAAR), the brain's main inhibitory neuroreceptor. When HEK293 cells transfected with OX1R and the α1, ß1, and γ2S subunits of GABAAR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the ß1 subunit of GABAAR, and the inhibition could itself be attenuated by (1) kinase inhibitors (of protein kinase C and CaM kinase II) and (2) the mutation, to alanine, of serine 409 of the ß1 subunit, a site previously identified in phosphorylation-dependent regulation in other pathways. These results are the first to directly support the feasibility of postsynaptic crosstalk between Ox-A and GABAAR, indicating a process in which Ox-A could promote phosphorylation of the ß1 subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABAAR crosstalk would cause the depolarising influence of Ox-A to be boosted, a type of positive feedback that could, for example, facilitate the ability to abruptly awake.

Pseudoaneurysm of homograft placed in right ventricular outflow tract.

Pseudoaneurysm of the right ventricular outflow tract after homograft placement is an infrequent complication after intracardiac repair for tetralogy of Fallot. We report two cases of pseudoaneurysm of right ventricular outflow tract after homograft placement for surgical repair of tetralogy of Fallot with pulmonary atresia.