Mutations in the KCNJ5 (potassium inwardly rectifying channel subfamily J member 5) gene, encoding an inwardly rectifying potassium channel, can drive aldosterone overproduction in a subset of aldosterone-producing adenomas and in familial hyperaldosteronism type III. Our objective was to identify small molecule compounds that specifically antagonize mutant KCNJ5 channels. Virtual screening of over 6 million small molecules identified compounds that putatively bind to KCNJ5 channels. The effect of 108 of these candidates was evaluated in vitro in human adrenocortical cells (HAC15) with inducible expression of wild-type or mutated forms of KCNJ5. Assessment encompassed cell viability, flow cytometry, gene expression, and adrenal steroid quantification via liquid chromatography-tandem mass spectrometry. Compounds antagonizing mutated KCNJ5 function were identified by evaluating their ability to rescue adrenal cell death induced by overexpression of mutant KCNJ5. A spiroquinoline compound, referred to as compound 81 (C81), effectively rescued cell death induced by KCNJ5 L168R in both monolayer and spheroid HAC15 cell cultures. C81 treatment caused a 69% to 85% reduction in CYP11B2 (aldosterone synthase) mRNA levels induced by KCNJ5 L168R, G151R, or T158A expression, compared with untreated cells. C81 also reduced aldosterone secretion by 65% in cells expressing KCNJ5 L168R and decreased 18-oxocortisol and 18-hydroxycortisol production by 78% and 90%, respectively. However, C81 had no significant effect on steroid secretion in cells overexpressing wild-type KCNJ5. C81 shows potential as a small molecule antagonist to specifically target pathological aldosterone secretion in familial hyperaldosteronism type III or KCNJ5-mutated aldosterone-producing adenomas. These findings suggest new avenues for genotype-based primary aldosteronism diagnostics and targeted treatments, contributing to personalized patient care.

Abnormalities in G-protein-gated inwardly rectifying potassium (GIRK) channels have been implicated in diseased states of the cardiovascular system; however, the role of GIRK4 (Kir3.4) in cardiac physiology and pathophysiology has yet to be completely understood. Within the heart, the KACh channel, consisting of two GIRK1 and two GIRK4 subunits, plays a major role in modulating the parasympathetic nervous system's influence on cardiac physiology. Being that GIRK4 is necessary for the functional KACh channel, KCNJ5, which encodes GIRK4, it presents as a therapeutic target for cardiovascular pathology. Human variants in KCNJ5 have been identified in familial hyperaldosteronism type III, long QT syndrome, atrial fibrillation, and sinus node dysfunction. Here, we explore the relevance of KCNJ5 in each of these diseases. Further, we address the limitations and complexities of discerning the role of KCNJ5 in cardiovascular pathophysiology, as identical human variants of KCNJ5 have been identified in several diseases with overlapping pathophysiology.

Pathogenic variants in KCNJ5, encoding the GIRK4 (Kir3.4) potassium channel, have been implicated in the pathogenesis of familial hyperaldosteronism type-III (FH-III) and sporadic primary aldosteronism (PA). In addition to aldosterone, glucocorticoids are often found elevated in PA in association with KCNJ5 pathogenic variants, albeit at subclinical levels. However, to date no GIRK4 defects have been linked to Cushing syndrome (CS). We present the case of a 10-year-old child who presented with CS at an early age due to bilateral adrenocortical hyperplasia (BAH). The patient was placed on low-dose ketoconazole (KZL), which controlled hypercortisolemia and CS-related signs. Discontinuation of KZL for even 6 weeks led to recurrent CS. Screening for known genes causing cortisol-producing BAHs (PRKAR1A, PRKACA, PRKACB, PDE11A, PDE8B, ARMC5) failed to identify any gene defects. Whole-exome sequencing showed a novel KCNJ5 pathogenic variant (c.506T>C, p.L169S) inherited from her father. In vitro studies showed that the p.L169S variant affects conductance of the Kir3.4 channel without affecting its expression or membrane localization. Although there were no effects on steroidogenesis in vitro, there were modest changes in protein kinase A activity. In silico analysis of the mutant channel proposed mechanisms for the altered conductance. We present a pediatric patient with CS due to BAH and a germline defect in KCNJ5. Molecular investigations of this KCNJ5 variant failed to show a definite cause of her CS. However, this KCNJ5 variant differed in its function from KCNJ5 defects leading to PA. We speculate that GIRK4 (Kir3.4) may play a role in early human adrenocortical development and zonation and participate in the pathogenesis of pediatric BAH.

Somatic variants in KCNJ5 are the most common cause of primary aldosteronism (PA). There are few patients with PA in whom the disease is caused by germline variants in the KCNJ5 potassium channel gene (familial hyperaldosteronism type III-FH-III). A 5-year-old patient who developed hypertension due to bilateral adrenocortical hyperplasia (BAH) causing PA had negative peripheral DNA testing for any known genetic causes of PA. He was treated medically with adequate control of his PA but by the third decade of his life, due to worsening renal function, he underwent bilateral adrenalectomy. Focused exome sequencing in multiple nodules of his BAH uncovered a "hot-spot" pathogenic KCNJ5 variant, while repeated Sanger sequencing showed no detectable DNA defects in peripheral blood and other tissues. However, whole exome, "deep" sequencing revealed that 0.23% of copies of germline DNA did in fact carry the same KCNJ5 variant that was present in the adrenocortical nodules, suggesting low level germline mosaicism for this PA-causing KCNJ5 defect. Thus, this patient represents a unique case of BAH due to a mosaic KCNJ5 defect. Undoubtedly, his milder PA compared with other known cases of FH-III, was due to his mosaicism. This case has a number of implications for the prognosis, treatment, and counseling of the many patients with PA due to BAH that are seen in hypertension clinics.

Less than 15% of hypertension cases in children are secondary to a primary hyperaldosteronism. This is idiopathic in 60% of the cases, secondary to a unilateral adenoma in 30% and 10% remaining by primary adrenal hyperplasia, familial hyperaldosteronism, ectopic aldosterone production or adrenocortical carcinoma.To date, four types of familial hyperaldosteronism (FH I to FH IV) have been reported. FH III is caused by germline mutations in KCNJ5, encoding the potassium channel Kir3.4. The mutations cause the channel to lose its selectivity for potassium, allowing large quantities of sodium to enter the cell. As a consequence, the membrane depolarizes, voltage-gated calcium channels open, calcium enters the cell, initiating the cascade that leads to aldosterone synthesis. Somatic mutations in KCNJ5 has also been described in aldosterone-producing adenomas. The most frequent presentation of FH III is with severe hyperaldosteronism symptoms and resistance to pharmacological therapy which leads to bilateral adrenalectomy. We will review current literature and describe a child with FH III due to a novel de novo deletion in KCNJ5 with wild phenotype as a sign of clinical variability of this disease.