title: A novel KCNJ5-insT149 somatic mutation close to, but outside, the selectivity filter causes resistant hypertension by loss of selectivity for potassium.
abstract: BACKGROUND
  Understanding the function of the KCNJ5 potassium channel through characterization of naturally occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism.
  
  
  OBJECTIVE
  We sought for such novel KCNJ5 channel mutations in a large database of patients with aldosterone-producing adenomas (APAs).
  
  
  METHODS
  We discovered a novel somatic c.446insAAC insertion, resulting in the mutant protein KCNJ5-insT149, in a patient with severe drug-resistant hypertension among 195 consecutive patients with a conclusive diagnosis of APA, 24.6% of whom showed somatic KCNJ5 mutations. By site-directed mutagenesis, we created the mutated cDNA that was transfected, along with KCNJ3 cDNA, in mammalian cells. We also localized CYP11B2 in the excised adrenal gland with immunohistochemistry and immunofluorescence using an antibody specific to human CYP11B2. Whole-cell patch clamp recordings, CYP11B2 mRNA, aldosterone measurement, and molecular modeling were performed to characterize the novel KCNJ5-insT149 mutation.
  
  
  RESULTS
  Compared with wild-type and mock-transfected adrenocortical cells, HAC15 cells expressing the mutant KCNJ5 showed increased CYP11B2 expression and aldosterone secretion. Mammalian cells expressing the mutated KCNJ5-insT149 channel exhibited a strong Na(+) inward current and, in parallel, a substantial rise in intracellular Ca(2+), caused by activation of voltage-gated Ca(2+) channels and reduced Ca(2+) elimination by Na(+)/Ca(2+) exchangers, as well as an increased production of aldosterone.
  
  
  CONCLUSIONS
  This novel mutation shows pathological Na(+) permeability, membrane depolarization, raised cytosolic Ca(2+), and increased aldosterone synthesis. Hence, a novel KCNJ5 channelopathy located after the pore α-helix preceding the selectivity filter causes constitutive secretion of aldosterone with ensuing resistant hypertension in a patient with a small APA.
authors: Department of Medicine-DIMED (M.K., B.C., L.L., F.G., G.P.R.), Department of Internal Medicine 4, and Department of Biomedical Sciences (G.Z.), University of Padua, 35126 Padua, Italy; Department of Medical Cell Biology (J.S., S.B., R.W.), University of Regensburg, 93053 Regensburg, Germany; Max Delbrück Center for Molecular Medicine (V.F., M.B.), 13092 Berlin, Germany; Division of Endocrinology (C.G.-S.), G. V. (Sonny) Montgomery Veterans Affairs Medical Center and Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi 39216.
authors: Maniselvan
authors: M
authors: Kuppusamy
authors: Brasilina
authors: B
authors: Caroccia
authors: Julia
authors: J
authors: Stindl
authors: Sascha
authors: S
authors: Bandulik
authors: Livia
authors: L
authors: Lenzini
authors: Francesca
authors: F
authors: Gioco
authors: Veniamin
authors: V
authors: Fishman
authors: Giuseppe
authors: G
authors: Zanotti
authors: Celso
authors: C
authors: Gomez-Sanchez
authors: Michael
authors: M
authors: Bader
authors: Richard
authors: R
authors: Warth
authors: Gian Paolo
authors: GP
authors: Rossi
mesh_terms: D000236:Adenoma / Q000235:genetics* / Q000378:metabolism; D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism; D018246:Adrenocortical Adenoma / Q000235:genetics* / Q000378:metabolism; D000368:Aged; D000450:Aldosterone / Q000378:metabolism; D000959:Antihypertensive Agents / Q000627:therapeutic use; D002460:Cell Line; D004351:Drug Resistance; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006929:Hyperaldosteronism / Q000235:genetics* / Q000378:metabolism; D006973:Hypertension / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D008297:Male; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism*
chemical_list: D000959:Antihypertensive Agents; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C484274:KCNJ5 protein, human; D000450:Aldosterone; D011188:Potassium