Hypertension with or without adrenal hyperplasia due to different inherited mutations in the potassium channel KCNJ5

Abstract

We recently implicated two recurrent somatic mutations in an adrenal potassium channel, KCNJ5, as a cause of aldosterone-producing adrenal adenomas (APAs) and one inherited KCNJ5 mutation in a Mendelian form of early severe hypertension with massive adrenal hyperplasia. The mutations identified all altered the channel selectivity filter, producing increased Na(+) conductance and membrane depolarization, the signal for aldosterone production and proliferation of adrenal glomerulosa cells. We report herein members of four kindreds with early onset primary aldosteronism of unknown cause. Sequencing of KCNJ5 revealed that affected members of two kindreds had KCNJ5(G151R) mutations, identical to one of the prevalent recurrent mutations in APAs. These individuals had severe progressive aldosteronism and hyperplasia requiring bilateral adrenalectomy in childhood for blood pressure control. Affected members of the other two kindreds had KCNJ5(G151E) mutations, which are not seen in APAs. These subjects had easily controlled hypertension and no evidence of hyperplasia. Surprisingly, electrophysiology of channels expressed in 293T cells demonstrated that KCNJ5(G151E) was the more extreme mutation, producing a much larger Na(+) conductance than KCNJ5(G151R), resulting in rapid Na(+)-dependent cell lethality. We infer that this increased lethality limits adrenocortical cell mass and the severity of aldosteronism in vivo, accounting for the milder phenotype among these patients. These findings demonstrate striking variations in phenotypes and clinical outcome resulting from different mutations of the same amino acid in KCNJ5 and have implications for the diagnosis and pathogenesis of primary aldosteronism with and without adrenal hyperplasia.

Fig. 1.

KCNJ5 mutations in patients with early onset primary aldosteronism. (Left) Pedigrees are shown, with affected subjects shown as filled symbols and unaffected family members as open symbols; subjects are numbered as in Table 1. The index case is denoted with an arrow. (Right) Sanger DNA sequence traces and corresponding amino acids at KCNJ5 positions 150–152 of tested subjects are shown. In each case, a single-base pair substitution leads to a missense mutation: G151R in affected members of K767 and K409 and G151E in families 1486 and 124. The mutation in subject 409-1 is a de novo mutation.

Fig. 2.

Survival of 293T cells transfected with WT and mutant KCNJ5 channels. WT or mutant eGFP-tagged KCNJ5 was transfected into 293T cells, and the percentage of eGFP-positive cells was measured at indicated times by flow cytometry (20,000 events counted per data point). (A) Incubation in physiologic medium (Materials and Methods) shows lower percentage of cells expressing KCNJ5^G151R than KCNJ5^WT at 36 h (P = 0.003) and lower percentage of cells expressing KCNJ5^G151E than either KCNJ5^WT (P < 0.0001) or KCNJ5^G151R (P = 0.003). (B) Reduction of extracellular Na⁺ by choline substitution significantly increased survival of cells expressing KCNJ5^G151R and KCNJ5^G151E at 24 and 36 h, with a more pronounced effect on the G151E mutant (for both mutant channels, P = 0.02 for low Na⁺ vs. normal medium at 24 h; P = 0.04 at 36 h).

Fig. 3.

KCNJ5^G151E channels have higher Na⁺ permeability than KCNJ5^G151R channels. Whole-cell currents of 293T cells transiently transfected with WT or mutant KCNJ5 channels were measured by using the patch-clamp technique and the voltage protocol indicated in A. Because incubation in solutions with physiological Na⁺ concentrations led to death of cells expressing KCNJ5^G151E, an extracellular solution containing 40 mM NaCl, 100 mM KCl, and 2 mM MgCl₂ (pH 7.4, adjusted with KOH) and an intracellular solution containing 140 mM KCl, 4 mM MgCl₂, and 5 mM Hepes (pH 7.4, adjusted with KOH) was used (control). In Ba²⁺ conditions, 1 mM Ba²⁺ was added to the above extracellular medium. In Ba²⁺/choline conditions, choline was substituted for Na⁺ as described in Materials and Methods. Representative whole-cell recordings are shown in A, and I–V curves are shown in B. In B, black, red, and green symbols denote control, Ba²⁺, and Ba²⁺/choline conditions, respectively. For better visibility, only one in two data points is plotted where curves overlap. Currents were negligible following transfection with empty vector (Fig. S3). Typical inwardly rectifying currents were observed for the WT channel; barium-sensitivity was abolished in the mutant channels. KCNJ5^G151E showed larger currents than KCNJ5^G151R, which were abolished by substitution of choline for Na⁺, indicating a predominant Na⁺ conductance. (C) Mean current amplitudes at −100 mV for WT and mutant channels using the solutions described in A. Net Na⁺ current is calculated by subtracting the current after choline substitution from the Ba²⁺ resistant current. Na⁺ current is significantly larger in the G151E mutant than in G151R mutant (P < 0.001). n ≥ 3 for each condition.