Novel Insertion Mutation in KCNJ5 Channel Produces Constitutive Aldosterone Release From H295R Cells.
Primary aldosteronism accounts for 5%-10% of hypertension and in a third of cases is caused by autonomous aldosterone production by adenomas (APA). Somatic mutations in the potassium channel encoded by KCNJ5 have been detected in surgically removed APAs. To better understand the role of these mutations, we resequenced the KCNJ5 channel in a large Australian primary aldosteronism cohort. KCNJ5 mutations were detected in 37 APAs (45% of the cohort), including previously reported E145Q (n = 3), G151R (n = 20), and L168R (n = 13) mutations. In addition, we found a novel 12-bp in-frame insertion mutation (c.414-425dupGCTTTCCTGTTC, A139_F142dup) that duplicates the AFLF sequence in the pore helix upstream of the selectivity filter. Expressed in Xenopus oocytes, the A139_F142dup mutation depolarized the oocytes and produced a G-protein-sensitive Na(+) current with altered K(+) selectivity and loss of inward rectification but retained Ba(2+) sensitivity. Transfected into H295R cells, A139_F142dup increased basal aldosterone release 2.3-fold over the wild type. This was not increased further by incubation with angiotensin II. Although the A139_F142dup mutant trafficked to the plasma membrane of H295R cells, it showed reduced tetramer stability and surface expression compared with the wild-type channel. This study confirms the frequency of somatic KCNJ5 mutations in APAs and the novel mutation identified (A139_F142dup) extend the phenotypic range of the known KCNJ5 APA mutations. Being located in the pore helix, it is upstream of the previously reported mutations and shares some features in common with selectivity filter mutants but additionally demonstrates insensitivity to angiotensin II and decreased channel stability.
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Iris
I
Hardege
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Shengxin
S
Xu
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Richard D
RD
Gordon
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Andrew J
AJ
Thompson
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Nichola
N
Figg
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Michael
M
Stowasser
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Ruth
R
Murrell-Lagnado
Divisions of Experimental Medicine and Immunotherapeutics (I.H., K.M.O.) and Cardiovascular Medicine (N.F.), Department of Medicine, and Department of Pharmacology (A.J.T., R.M.-L.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; and Endocrine Hypertension Research Centre (S.X., R.D.G., M.S.), University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane 4072, Australia.
Kevin M
KM
O'Shaughnessy
D000450:Aldosterone / Q000378:metabolism*; D000595:Amino Acid Sequence; D000809:Angiotensins / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D055724:Electrophysiological Phenomena / Q000187:drug effects; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000187:drug effects; D009154:Mutation / Q000235:genetics*; D055550:Protein Stability / Q000187:drug effects; D014981:Xenopus
D000809:Angiotensins; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C484274:KCNJ5 protein, human; D000450:Aldosterone