Abstract 011: Identification and Electrophysiological Characterization of a Novel Somatic Mutation (insT149KCNJ5) of the Potassium Channel Kir3.4 (KCNJ5)
Context: Understanding the function of the Kir3.4 (KCNJ5 gene) potassium channel through characterization of occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism.
Objective: To identify novel KCNJ5 channel mutations and functionally characterize them in a large database of patients with aldosterone-producing adenomas (APA).
Methods: We sequenced the APA and germinal DNA of 195 consecutive patients, diagnosed with the four corners criteria of the PAPY study. Among the 24.6% (48/195) APA patients who showed somatic KCNJ5 mutations we discovered a novel c.446insAAC insertion resulting in the mutant protein KCNJ5-insT149 in a patient with severe drug-resistant hypertension. The mutated cDNA generated by site-directed mutagenesis was transfected along with KCNJ3 cDNA in mammalian cells. 17α-hydroxylase, CYP11B1, and CYP11B2 were immunochemically localized in the excised adrenal gland. Whole-cell patch clamp recordings, CYP11B2 mRNA, aldosterone and intracellular Ca2+ measurement (Fura-2), and molecular modeling were performed to characterize the KCNJ5-insT149 mutation.
Results: The patient’s high blood pressure was long-term cured; his LVMI fell from 168 to 106 g/m2 after 2 years follow-up. Compared to wild type and mock-transfected HAC15 adrenocortical cells, those expressing the mutant KCNJ5 showed increased CYP11B2 expression (expression fold change: 2.9±0.3, p<0.05 vs mock transfected cells) and aldosterone secretion (260 pg/μg RNA, p<0.05 vs mock cells). The HEK293 cells expressing the mutated KCNJ5-insT149 channel exhibited a strong Na+ inward current, and a substantial rise in intracellular Ca2+. The L-type Ca2+ channel blocker verapamil [10 μM] inhibited by 50% the pathological Na+ inward current, while both the Na+/Ca2+ exchanger blocker KB-R7943 [10 μM] and the removal of extracellular Na+ abolished it.
Conclusions: We identified a novel mutation of the Kir3.4 channelopathy located after the pore α-helix preceding the selectivity filter, which causes pathological Na+ permeability, membrane depolarization, raised cytosolic Ca2+, and constitutive hypersecretion of aldosterone resulting in ensuing pseudo resistant hypertension.
Author Disclosures: G. Rossi: None. M. Kuppusamy: None. B. Caroccia: None. M. Bader: None. L. Lenzini: None. J. Stindl: None. S. Bandulik: None. F. Gioco: None. V. Fishman: None. G. Zanotti: None. C. Gomez-Sanchez: None. R. Warth: None.
- © 2014 by American Heart Association, Inc.