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 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 β-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The β-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.

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.

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UNASSIGNED: The prevalence of unilateral primary aldosteronism (UPA) with cortisol co-secretion varies geographically.
UNASSIGNED: To investigate the prevalence and clinical characteristics of UPA with cortisol co-secretion in a Chinese population.
UNASSIGNED: Retrospective cohort study.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

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 PKCβ. 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 PKCβ 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.

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.

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.

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.