K+ channel mutations in adrenal aldosterone-producing adenomas and hereditary hypertension

Abstract

Endocrine tumors such as aldosterone-producing adrenal adenomas (APAs), a cause of severe hypertension, feature constitutive hormone production and unrestrained cell proliferation; the mechanisms linking these events are unknown. We identify two recurrent somatic mutations in and near the selectivity filter of the potassium (K(+)) channel KCNJ5 that are present in 8 of 22 human APAs studied. Both produce increased sodium (Na(+)) conductance and cell depolarization, which in adrenal glomerulosa cells produces calcium (Ca(2+)) entry, the signal for aldosterone production and cell proliferation. Similarly, we identify an inherited KCNJ5 mutation that produces increased Na(+) conductance in a Mendelian form of severe aldosteronism and massive bilateral adrenal hyperplasia. These findings explain pathogenesis in a subset of patients with severe hypertension and implicate loss of K(+) channel selectivity in constitutive cell proliferation and hormone production.

Fig. 1

Mutations in KCNJ5 in aldosterone-producing adenoma and inherited aldosteronism. (A) Sequences of blood and tumor genomic DNA and tumor cDNA of KCNJ5 codons 150 to 152 in APA12. (B) Sequences of KCNJ5 codons 167 to 169 in APA15. (C) KCNJ5 mutation in kindred HPA1. At top, kindred structure is shown; affected members are shown as filled symbols; gray symbol represents a subject who died at age 36 with severe hypertension, suspected to be affected. KCNJ5 sequences of codons 157 to 159 are shown. Reverse strand traces for (A) to (C) are shown in fig. S4. (D) Conservation of G151, T158, and L168 in orthologs and paralogs. These positions are conserved among chordate orthologs that last shared a common ancestor 750 million years ago. H.s., Homo sapiens; M.m., Mus musculus; G.g., Gallus gallus; X.t., Xenopus tropicalis; D.r., Danio rerio; C.i., Ciona intestinalis. Shown below are the sequences of selected human inward rectifier K⁺ channels, demonstrating high conservation among diverse members of this family.

Fig. 2

Location of human mutations in KCNJ5 mapped onto the crystal structure of chicken K⁺ channel KCNJ12 (16). (A) Location of mutations. The extracellular and transmembrane domains of two subunits from the channel tetramer are shown with K⁺ ions (purple) traversing the selectivity filter; human KCNJ5 and chicken KCNJ12 are 89% identical in the pore helix and selectivity filter. G151 lies in the selectivity filter at a position conserved among virtually all K⁺ channels. Its main chain carbonyl group faces the channel pore. T158 lies just above the selectivity filter, and L168 is in the second transmembrane domain (inner helix) with its side chain projecting toward the selectivity filter. (B) View of the side chains of L168 and the highly conserved Y152 of the selectivity filter, showing their close proximity. (C) View of T158, which makes hydrogen bonds with conserved positions P128 and C129.

Fig. 3

KCNJ5 mutations result in loss of channel selectivity and membrane depolarization. (A) Representative whole-cell recordings of 293T cells transfected with empty vector or KCNJ3 plus WT or mutant KCNJ5. The pipette holding potential was 0 mV before clamping, and the cell was clamped from −100mV to +60 mV, with 20 mV increments. Top row: extracellular solution contained 140 mM NaCl, 5 mM KCl, 1.8 mM MgCl₂, 1.8 mM CaCl₂, 10 mM HEPES, pH 7.4; intracellular solution contained 140 mM KCl, 4 mM MgCl₂, 1 mM CaCl₂, 1 mM EGTA, 5 mM HEPES, pH 7.4. Middle row: 1mM BaCl₂ was added. Bottom row: 140 mM choline chloride was substituted for extracellular NaCl. (B) Current-voltage relationships from cells expressing indicated constructs (n = 3 to 7 for each construct). Reversal potentials in control conditions are indicated. WT channel shows a highly negative reversal potential and is inhibited by Ba²⁺ but not substitution of choline for Na⁺ (see also fig. S7). Mutant channels show less negative reversal potentials; currents are inhibited by elimination of Na⁺ but show variable inhibition by Ba²⁺. (C) K⁺:Na⁺ permeability ratios calculated from the reversal potentials (11) show loss of ion selectivity of the mutant channels. Data in (B) and (C) are shown as mean ± SEM. Reversal potentials and K⁺:Na⁺ permeability ratios are significantly different between wild-type and mutant channels (P < 0.01 by Student’s t test).

Fig. 4

Proposed mechanism underlying aldosterone-producing adenoma and Mendelian aldosteronism. (A) Adrenal glomerulosa cells have a high resting K⁺ conductance, which produces a highly negative membrane potential (2). (B) Membrane depolarization by either elevation of extracellular K⁺ or closure of K⁺ channels by angiotensin II activates voltage-gated Ca²⁺ channels, increasing intracellular Ca²⁺ levels (1). This provides signals for increased expression of enzymes required for aldosterone biosynthesis, such as aldosterone synthase, and for increased cell proliferation. (C) Channels containing KCNJ5 with G151R, T158A, or L168R mutations conduct Na⁺, resulting in Na⁺ entry, chronic depolarization, constitutive aldosterone production, and cell proliferation.