Domain-selective and sex-dependent regulation of learning and memory in mice by GIRK channel activity in CA1 pyramidal neurons of the dorsal hippocampus.

G protein-gated inwardly rectifying K+ (GIRK) channels mediate the postsynaptic inhibitory effect of many neurotransmitters in the hippocampus and are implicated in neurological disorders characterized by cognitive deficits. Here, we show that enhancement or suppression of GIRK channel activity in dorsal CA1 pyramidal neurons disrupted novel object recognition in mice, without impacting open field activity or avoidance behavior. Contextual fear learning was also unaffected, but extinction of contextual fear was disrupted by suppression of GIRK channel activity in male mice. Thus, the strength of GIRK channel activity in dorsal CA1 pyramidal neurons regulates select cognitive task performance in mice.

Amyloid-ß oligomers trigger sex-dependent inhibition of GIRK channel activity in hippocampal neurons in mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques and cognitive decline, the latter of which is thought to be driven by soluble oligomeric amyloid-ß (oAß). The dysregulation of G protein-gated inwardly rectifying K+ (GIRK; also known as Kir3) channels has been implicated in rodent models of AD. Here, seeking mechanistic insights, we uncovered a sex-dependent facet of GIRK-dependent signaling in AD-related amyloid pathophysiology. Synthetic oAß1-42 suppressed GIRK-dependent signaling in hippocampal neurons from male mice, but not from female mice. This effect required cellular prion protein, the receptor mGluR5, and production of arachidonic acid by the phospholipase PLA2. Although oAß suppressed GIRK channel activity only in male hippocampal neurons, intrahippocampal infusion of oAß or genetic suppression of GIRK channel activity in hippocampal pyramidal neurons impaired performance on a memory test in both male and female mice. Moreover, genetic enhancement of GIRK channel activity in hippocampal pyramidal neurons blocked oAß-induced cognitive impairment in both male and female mice. In APP/PS1 AD model mice, GIRK-dependent signaling was diminished in hippocampal CA1 pyramidal neurons from only male mice before cognitive deficit was detected. However, enhancing GIRK channel activity rescued cognitive deficits in older APP/PS1 mice of both sexes. Thus, whereas diminished GIRK channel activity contributes to cognitive deficits in male mice with increased oAß burden, enhancing its activity may have therapeutic potential for both sexes.

Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay.

The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the ß-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The ß-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid's GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.

[Research progress of KCNJ5 gene in aldosterone-producing adenoma].

Aldosterone-producing adenoma is a subtype of primary aldosteronism. Recent advancements in multi-omics research have led to significant progress in understanding primary aldosteronism at the genetic level. Among the various genes associated with the development of aldosterone-producing adenomas, the KCNJ5 (potassium inwardly rectifying channel, subfamily J, member 5) gene has received considerable attention due to its prevalence as the most common somatic mutation gene in primary aldosteronism. This paper aims to integrate the existing evidence on the involvement of KCNJ5 gene in the pathogenesis of aldosterone-producing adenomas, to enhance the understanding of the underlying mechanisms of aldosterone-producing adenomas from the perspective of genetics, and to provide novel insights for the clinical diagnosis and treatment of aldosterone-producing adenomas.

The Role of KACh Channels in Atrial Fibrillation.

This manuscript explores the intricate role of acetylcholine-activated inward rectifier potassium (KACh) channels in the pathogenesis of atrial fibrillation (AF), a common cardiac arrhythmia. It delves into the molecular and cellular mechanisms that underpin AF, emphasizing the vital function of KACh channels in modulating the atrial action potential and facilitating arrhythmogenic conditions. This study underscores the dual nature of KACh activation and its genetic regulation, revealing that specific variations in potassium channel genes, such as Kir3.4 and K2P3.1, significantly influence the electrophysiological remodeling associated with AF. Furthermore, this manuscript identifies the crucial role of the KACh-mediated current, IKACh, in sustaining arrhythmia through facilitating shorter re-entry circuits and stabilizing the re-entrant circuits, particularly in response to vagal nerve stimulation. Experimental findings from animal models, which could not induce AF in the absence of muscarinic activation, highlight the dependency of AF induction on KACh channel activity. This is complemented by discussions on therapeutic interventions, where KACh channel blockers have shown promise in AF management. Additionally, this study discusses the broader implications of KACh channel behavior, including its ubiquitous presence across different cardiac regions and species, contributing to a comprehensive understanding of AF dynamics. The implications of these findings are profound, suggesting that targeting KACh channels might offer new therapeutic avenues for AF treatment, particularly in cases resistant to conventional approaches. By integrating genetic, cellular, and pharmacological perspectives, this manuscript offers a holistic view of the potential mechanisms and therapeutic targets in AF, making a significant contribution to the field of cardiac arrhythmia research.

Double CYP11B1/CYP11B2 Immunohistochemistry and Detection of KCNJ5 Mutations in Primary Aldosteronism.

CONTEXT: The search for somatic mutations in adrenals resected from patients with primary aldosteronism (PA) is performed by Sanger sequencing, often implemented with immunohistochemistry (IHC)-guidance focused on aldosterone-producing (CYP11B2-positive) areas. OBJECTIVE: To investigate the impact of double IHC for CYP11B1 and CYP11B2 on Sanger and next-generation sequencing (NGS). METHODS: We investigated 127 consecutive adrenal aldosterone-producing adenomas from consenting surgically cured PA patients using double IHC for CYP11B1 and CYP11B2, by Sanger sequencing and NGS. RESULTS: Double IHC for CYP11B2 and CYP11B1 revealed 3 distinct patterns: CYP11B2-positive adenoma (pattern 1), mixed CYP11B1/CYP11B2-positive adenoma (pattern 2), and adrenals with multiple small CYP11B2-positive nodules (pattern 3). Sanger sequencing allowed detection of KCNJ5 mutations in 44% of the adrenals; NGS revealed such mutations in 10% of those negative at Sanger and additional mutations in 61% of the cases. Importantly the rate of KCNJ5 mutations differed across patterns: 17.8% in pattern 1, 71.4% in pattern 2, and 10.7% in pattern 3 (χ2 = 22.492, P < .001). CONCLUSION: NGS allowed detection of mutations in many adrenals that tested negative at Sanger sequencing. Moreover, the different distribution of KCNJ5 mutations across IHC patterns indicates that IHC-guided sequencing protocols selecting CYP11B2-positive areas could furnish results that might not be representative of the entire mutational status of the excised adrenal, which is important at a time when KCNJ5 mutations are suggested to drive management of patients with aldosterone-producing adenomas.

The influence of cortisol co-secretion on clinical characteristics and postoperative outcomes in unilateral primary aldosteronism.

Context: The prevalence of unilateral primary aldosteronism (UPA) with cortisol co-secretion varies geographically. Objective: To investigate the prevalence and clinical characteristics of UPA with cortisol co-secretion in a Chinese population. Design: Retrospective cohort study. Methods: We recruited 580 patients with UPA who underwent cosyntropin stimulation test (CST) after the 1-mg dexamethasone suppression test (DST) and retrospectively analyzed the clinical characteristics and postoperative outcomes of UPA with and without cortisol co-secretion. Results: UPA with cortisol co-secretion (1 mg DST>1.8 ug/dL) was identified in 65 of 580 (11.2%) patients. These patients were characterized by older age, longer duration of hypertension, higher concentration of plasma aldosterone and midnight cortisol, lower adrenocorticotropic hormone (ACTH) and dehydroepiandrosterone sulfate (DHEAS), larger tumor diameter, and more history of diabetes mellitus. Cortisol and aldosterone levels were higher and DHEAS level was lower in UPA with cortisol co-secretion at 0-120 min after CST. Among 342 UPA patients with KCNJ5 gene sequencing and follow-up results, the complete clinical success rate was lower in UPA with cortisol co-secretion (33.3% vs. 56.4%, P<0.05); the complete biochemical success rate and KCNJ5 mutation did not differ between the two groups. Age, tumor size, and ACTH were independent predictors of UPA with cortisol co-secretion. Sex, BMI, duration of hypertension, KCNJ5 mutation, and cortisol co-secretion were independent predictors for complete clinical success in UPA after surgery. Conclusions: UPA with cortisol co-secretion is not uncommon in China, but the clinical features were distinctly different from those without co-secretion. Cortisol co-secretion is an independent risk factor for incomplete clinical success after surgery in UPA.

Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCß. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCß do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

Cardiovascular Outcomes of KCNJ5 Mutated Aldosterone-Producing Adenoma: A Systematic Review.

BACKGROUND: While clinical features of KCNJ5-mutated aldosterone-producing adenoma (APA) have been reported, evidence of its clinical outcomes is lacking. We aimed to synthesize available literature about the associations between KCNJ5 mutation with cardiovascular and metabolic outcomes among patients with APA. METHODS: In this systematic review of observational studies, MEDLINE and Embase were searched through August 2022. Two independent authors screened the search results and extracted data from eligible observational studies investigating cardiovascular or metabolic outcomes between KCNJ5-mutated APAs and KCNJ5-non-mutated APAs. Risk of Bias In Non-randomized Studies of Interventions was used to assess the quality of the included studies. RESULTS: A total of 573 titles/abstracts were screened and after the expert opinion of the literature, full text was read in 20 titles/abstracts, of which 12 studies were included. Across 3 studies comparing the baseline or change in the cardiac function between KCNJ5-mutated APAs and KCNJ5-non-mutated APAs, all studies reported the association between impaired cardiac functions and KCNJ5 mutation status. Among 6 studies evaluating the cure of hypertension after surgery, all studies showed that KCNJ5 mutation was significantly associated with the cure of hypertension. In quality assessment, 7 studies were at serious risk of bias, while the remaining studies were at moderate risk of bias. CONCLUSIONS: This systematic review provided evidence of the significant association between KCNJ5 mutation and unfavorable cardiovascular outcomes in patients with primary aldosteronism. Further research is needed to improve the quality of evidence on this topic and elucidate the underlying mechanisms of the potential burden of KCNJ5 mutation.

Protocol for kinetic mode potassium channel assays on common plate readers and microscopes.

Fluorescence-based potassium channel assays are typically run on expensive, hard to obtain, fluorescence imaging kinetic plate readers that are uncommon in most laboratories. Here we describe the use of the Brilliant Thallium Snapshot assay to conduct an endpoint potassium channel assay, so that it can be used across multiple plate reader platforms that are more common in many labs. These methods will allow users to identify modulators of potassium channels. For this work, we have taken a kinetic mode Molecular Devices FLIPR based protocol and adapted it to be utilized on endpoint plate readers, such as the BMG Labtech PHERAstar, to identify activators of GIRK channels in CHO cells. We demonstrate that both plate readers are functionally competent at generating excellent Z' values which makes them ideally suited to finding corollary hits from the Sigma LOPAC 1,280 screening collection. Importantly, this assay has also been validated using a high content reader, demonstrating the possibility of spatially resolving signals from individual cells within a mixed cell population. The compendium of these results shows the flexibility, accessibility and functionality of endpoint-compatible potassium channel assay readouts on more common plate readers.

Structural determinants of the direct inhibition of GIRK channels by Sigma-1 receptor antagonist.

G-protein-gated inward rectifier K+ (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of the multifunctional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the regulation of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047, as well as BD1063, directly inhibited GIRK currents even in the absence of S1R and in a voltage-independent manner. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and identify the structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited GIRK channel currents, producing a much stronger inhibition of GIRK4 compared to GIRK2. It also inhibited ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combined with molecular docking analysis demonstrated the importance of Leu77 and Leu84 within the cytoplasmic, proximal N-terminal region and Glu147 within the pore-forming region of GIRK4 for inhibition by BD1047. The activator of GIRK channels, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4-associated diseases.

Insights into Activation Dynamics and Functional Sites of Inwardly Rectifying Potassium Channel Kir3.2 by an Elastic Network Model Combined with Perturbation Methods.

The inwardly rectifying potassium channel Kir3.2, a member of the inward rectifier potassium (Kir) channel family, exerts important biological functions through transporting potassium ions outside of the cell, during which a large-scale synergistic movement occurs among its different domains. Currently, it is not fully understood how the binding of the ligand to the Kir3.2 channel leads to the structural changes and which key residues are responsible for the channel gating and allosteric dynamics. Here, we construct the Gaussian network model (GNM) of the Kir3.2 channel with the secondary structure and covalent interaction information considered (sscGNM), which shows a better performance in reproducing the channel's flexibility compared with the traditional GNM. In addition, the sscANM-based perturbation method is used to simulate the channel's conformational transition caused by the activator PIP2's binding. By applying certain forces to the PIP2 binding pocket, the coarse-grained calculations generate the similar conformational changes to the experimental observation, suggesting that the topology structure as well as PIP2 binding are crucial to the allosteric activation of the Kir3.2 channel. We also utilize the sscGNM-based thermodynamic cycle method developed by us to identify the key residues whose mutations significantly alter the channel's binding free energy with PIP2. We identify not only the residues important for the specific binding but also the ones critical for the allosteric transition coupled with PIP2 binding. This study is helpful for understanding the working mechanism of Kir3.2 channels and can provide important information for related drug design.

Upregulated GIRK2 Counteracts Ethanol-Induced Changes in Excitability and Respiration in Human Neurons.

Genome-wide association studies (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified noncoding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G-protein-coupled inwardly rectifying potassium channel that regulates neuronal excitability. We studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multielectrode arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 d of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-induced changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons.

Leptin excites basolateral amygdala principal neurons and reduces food intake by LepRb-JAK2-PI3K-dependent depression of GIRK channels.

Leptin is an adipocyte-derived hormone that modulates food intake, energy balance, neuroendocrine status, thermogenesis, and cognition. Whereas a high density of leptin receptors has been detected in the basolateral amygdala (BLA) neurons, the physiological functions of leptin in the BLA have not been determined yet. We found that application of leptin excited BLA principal neurons by activation of the long form leptin receptor, LepRb. The LepRb-elicited excitation of BLA neurons was mediated by depression of the G protein-activated inwardly rectifying potassium (GIRK) channels. Janus Kinase 2 (JAK2) and phosphoinositide 3-kinase (PI3K) were required for leptin-induced excitation of BLA neurons and depression of GIRK channels. Microinjection of leptin into the BLA reduced food intake via activation of LepRb, JAK2, and PI3K. Our results may provide a cellular and molecular mechanism to explain the physiological roles of leptin in vivo.

Licorice metabolite 18ß-glycyrrhetinic acid activates G protein-gated inwardly rectifying K+ channels.

BACKGROUND AND PURPOSE: Licorice (liquorice) is a common food additive and is used in Chinese medicine. Excess licorice intake can induce atrial fibrillation. Patients with atrial fibrillation possess constitutively activated G protein-gated inwardly rectifying K+ (GIRK) channels. Whether licorice affects GIRK channel activity is unknown. We aimed to clarify the effects of licorice ingredients on GIRK current and the mechanism of action. EXPERIMENTAL APPROACH: A major component of licorice, glycyrrhizic acid (GA), and its metabolite, 18ß-glycyrrhetinic acid (18ß-GA), were tested. We performed electrophysiological recordings in Xenopus oocytes to examine the effects of GA and 18ß-GA on various GIRK subunits (Kir 3.1-Kir 3.4), mutagenesis analyses to identify the crucial residues for drug action and motion analysis in cultured rat atrial myocytes to clarify effects of 18ß-GA on atrial functions. KEY RESULTS: GA inhibited Kir 3.1-containing channels, while 18ß-GA activated all Kir 3.x subunits. A pore helix residue Phe137 in Kir 3.1 was critical for GA-mediated inhibition, and the corresponding Ser148 in Kir 3.2 was critical for 18ß-GA-mediated activation. 18ß-GA activated GIRK channel in a Gßγ -independent manner, whereas phosphatidylinositol 4,5-bisphosphate (PIP2 ) was essential for activation. Glu236 located at the cytoplasmic pore of Kir 3.2 appeared to be important to interactions with 18ß-GA. In rat atrial myocytes, 18ß-GA suppressed spontaneous beating via activation of GIRK channels. CONCLUSION AND IMPLICATIONS: GA acts as a novel GIRK inhibitor, and 18ß-GA acts as a novel GIRK activator. 18ß-GA alters atrial function via activation of GIRK channels. This study elucidates the pharmacological activity of licorice ingredients and provides information for drug design.

Single-Nucleus Analysis Reveals Tumor Heterogeneity of Aldosterone-Producing Adenoma.

BACKGROUND: Recent advances in omics techniques have allowed detailed genetic characterization of aldosterone-producing adenoma (APA). The pathogenesis of APA is characterized by tumorigenesis-associated aldosterone synthesis. The pathophysiological intricacies of APAs have not yet been elucidated at the level of individual cells. Therefore, a single-cell level analysis is speculated to be valuable in studying the differentiation process of APA. METHODS: We conducted single-nucleus RNA sequencing of APAs with KCNJ5 mutation and nonfunctional adenomas obtained from 3 and 2 patients, respectively. RESULTS: The single-nucleus RNA sequencing revealed the intratumoral heterogeneity of APA and identified cell populations consisting of a shared cluster of nonfunctional adenoma and APA. In addition, we extracted 2 cell fates in APA and obtained a cell population specialized in aldosterone synthesis. Genes related to ribosomes and neurodegenerative diseases were upregulated in 1 of these fates, whereas those related to the regulation of glycolysis were upregulated in the other fate. Furthermore, the total RNA reads in the nucleus were higher in hormonally activated clusters, indicating a marked activation of transcription per cell. CONCLUSIONS: The single-nucleus RNA sequencing revealed intratumoral heterogeneity of APA with KCNJ5 mutation. The observation of 2 cell fates in KCNJ5-mutated APAs provides the postulation that a heterogeneous process of cellular differentiation was implicated in the pathophysiological mechanisms underlying APA tumors.

De novo variants in KCNJ3 are associated with early-onset epilepsy.

BACKGROUND: KCNJ3 encodes a subunit of G-protein-coupled inwardly rectifying potassium channels, which are important for cellular excitability and inhibitory neurotransmission. However, the genetic basis of KCNJ3 in epilepsy has not been determined. This study aimed to identify the pathogenic KCNJ3 variants in patients with epilepsy. METHODS: Trio exome sequencing was performed to determine potential variants of epilepsy. Individuals with KCNJ3 variants were recruited for this study. Detailed clinical information and genetic data were obtained and systematically reviewed. Whole-cell patch-clamp recordings were performed to evaluate the functional consequences of the identified variants. RESULTS: Two de novo missense variants (c.998T>C (p.Leu333Ser) and c.938G>A (p. Arg313Gln)) in KCNJ3 were identified in two unrelated families with epilepsy. The variants were absent from the gnomAD database and were assumed to be damaging or probably damaging using multiple bioinformatics tools. They were both located in the C-terminal domain. The amino acid residues were highly conserved among various species. Clinically, the seizures occurred at a young age and were under control after combined treatment. Electrophysiological analysis revealed that the KCNJ3 Leu333Ser and Arg313Gln variants significantly compromised the current activities and exhibited loss-of-function (LOF) effects. CONCLUSION: Our findings suggest that de novo LOF variants in KCNJ3 are associated with early-onset epilepsy. Genetic testing of KCNJ3 in patients with epilepsy may serve as a strategy for precision medicine.

Urine steroid metabolomics as a diagnostic tool in primary aldosteronism.

Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.

Structural insights in the permeation mechanism of an activated GIRK2 channel.

G protein-gated inwardly rectifying potassium (GIRK) channels play a significant role in physiopathology by the regulation of cell excitability. This regulation depends on the K+ ion conduction induced by structural constrictions: the selectivity filters (SFs), helix bundle crossings (HBCs), and G-loop gates. To explore why no permeation occurred when the constrictions were kept in the open state, a 4-K+-related occupancy mechanism was proposed. Unfortunately, this hypothesis was neither assessed, nor was the energetic characteristics presented. To identify the permeation mechanism on an atomic level, all-atom molecular dynamic (MD) simulations and a coupled quantum mechanics and molecular mechanics (QM/MM) method were used for the GIRK2 mutant R201A. It was found that the R201A had a moderate conductive capability in the presence of PIP2. Furthermore, the 4-K+ group of ions was found to dominate the conduction through the activated HBC gate. This shielding-like mechanism was assessed by the potential energy barrier along the conduction pathway. Mutation studies did further support the assumption that E152 was responsible for the mechanism. Moreover, E152 was most probably facilitating the inflow of ions from the SF to the cavity. On the contrary, N184 had no remarkable effect on this mechanism, except for the conduction efficiency. These findings highlighted the necessity of a multi-ion distribution for the conduction to take place, and indicated that the K+ migration was not only determined by the channel conductive state in the GIRK channel. The here presented multi-ion permeation mechanism may help to provide an effective way to regulate the channelopathies.