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Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma
Ute I. Scholl, … , Wenhui Wang, Richard P. Lifton
Ute I. Scholl, … , Wenhui Wang, Richard P. Lifton
Published June 12, 2017
Citation Information: J Clin Invest. 2017;127(7):2739-2750. https://doi.org/10.1172/JCI91733.
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Research Article Endocrinology Nephrology

Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma

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Abstract

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

Authors

Ute I. Scholl, Laura Abriola, Chengbiao Zhang, Esther N. Reimer, Mark Plummer, Barbara I. Kazmierczak, Junhui Zhang, Denton Hoyer, Jane S. Merkel, Wenhui Wang, Richard P. Lifton

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Figure 1

Establishment of screening assays for KCNJ5MUT and KCNJ5WT inhibition.

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Establishment of screening assays for KCNJ5MUT and KCNJ5WT inhibition.
(...
(A) Reduced ATP levels following induction of KCNJ5G151R or KCNJ5L168R, but not KCNJ5WT, in HEK293 cells. Cell survival was measured using an ATP-based viability assay with luminescence as a read-out in stable inducible cell lines in the uninduced and induced states. Whereas induction of KCNJ5WT expression had no significant effect on ATP levels, induction of KCNJ5G151R and KCNJ5L168R led to a significant decrease in ATP levels. P values represent 2-tailed unpaired Student’s t test comparing uninduced vs. induced for each line. n = 8 for each condition, 2,000 cells per well (WT and G151R) or 4,000 cells per well (L168R). Bars represent mean, and error bars represent SD. (B) Evaluation of the KCNJ5WT membrane potential assay using BaCl2 using a standard curve of BaCl2 concentrations. Depolarization caused by increasing BaCl2 concentrations leads to an increase in fluorescence. Relative fluorescence at 2 mM BaCl2 was defined as 100%. Data were fitted with a 4-parameter nonlinear regression of log-dose versus response. n = 6. Dots represent mean, error bars represent SD.

Copyright © 2022 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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