Mutated KCNJ5 activates the acute and chronic regulatory steps in aldosterone production

  1. Tobias Else1⇑
  1. 1Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, Michigan, USA
  2. 2Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA
  3. 3Center for Arrhythmia Research, University of Michigan, Ann Arbor, Michigan, USA
  4. 4G. V. (Sonny) Montgomery VA Medical Center and Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi, USA
  5. 5Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA
  1. Correspondence should be addressed to T Else; Email: telse{at}umich.edu
  1. Figure 1
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    Figure 1

    Doxycycline causes a time-dependent induction of (A) KCNJ5 mRNA and (B) KCNJ5 protein in HAC15-TRE-KCNJ5T158A cells. Cells were treated with 1 μg/mL doxycycline for the indicated times. Quantitative RT-PCR was used for mRNA transcript detection, and western analyses for protein quantification. Doxycycline incubation caused a time-dependent increase in KCNJ5T158A mRNA (Panel A) and protein (Panel B). Results represent the mean±s.e.m. of at least five independent experiments for mRNA (Panel A) and three independent westerns (Panel B) (*P<0.05 vs basal).

  2. View larger version:
    Figure 2

    Electrophysiological analyses of cells expressing Kir3.4T158A or Kir3.4WT. Representative whole-cell currents of cells expressing Kir3.4T158A (by doxycycline treatment) or Kir3.4WT (n=5–6 per group) were measured under basal conditions (normal Tyrode’s solution), blockade of K+ conductance (through addition of 1 mM barium chloride) and in Na+-free conditions (replacing NaCl with choline chloride, in addition to K+ blockade). Panel A includes representative plots of current in a voltage-step protocol in the above-mentioned conditions. Panels B and C indicate the sensitivity of the Kir3.4T158A and Kir3.4WT channels to barium and choline. All current recordings are normalized to cell capacitance. The figure is available in colour online at http://dx.doi.org/10.1530/JME-15-0324

  3. Figure 3
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    Figure 3

    KCNJ5T158A increases CYP11B2 mRNA and protein expression, and aldosterone production through calcium-mediated pathway. Cells were incubated in 1 μg/mL doxycycline at indicated times, causing a time-dependent increase in CYP11B2 mRNA (Panel A), CYP11B2 protein (Panel B) and aldosterone production (Panel C). Pre-incubation with calcium channel blocker, verapamil (1, 3, 10 and 30 μM), caused a dose-dependent inhibition in KCNJ5T158A- and Ang II-stimulated aldosterone production (Panel D). Pretreatment with verapamil (10 μM) also inhibited KCNJ5T158A- and Ang II-stimulated CYP11B2 mRNA and protein expression (Panels E and F). Results represent the mean±s.e.m. of at least five independent experiments for mRNA (Panels A and E) and three independent westerns (Panels B and F). Black and gray bars represent the absence and presence of verapamil (10 μM), respectively. Statistical analyses were performed using one-way ANOVA (*P<0.05 vs basal, $P<0.05 vs corresponding treatment in the absence of verapamil, #P<0.05 vs corresponding treatment inthe absence of verapamil for panels D-F, $P<0.05 vs basal).

  4. Figure 4
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    Figure 4

    KCNJ5T158A effects on adrenal steroidogenesis by LC-MS/MS. A schematic representation of the steroidogenic pathway for aldosterone, cortisol and hybrid steroids in shown in Panel A. Analyses of steroids by LC-MS/MS (Panel B) indicates an increase in KCNJ5T158A-mediated synthesis (by 60 h doxycycline treatment) of aldosterone, its precursors (DOC and corticosterone), as well as in cortisol (F), 18OHF and 18oxoF. Black and gray bars represent the absence and presence of verapamil (10 μM), respectively. Statistical analyses were performed using one-way ANOVA (*P<0.05 vs basal, $P<0.05 vs corresponding treatment in the absence of verapamil).

  5. Figure 5
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    Figure 5

    KCNJ5T158A increases the activation of transcription factors required for CYP11B2 transcription, including NURR1 (A and B) and ATF2 (C). Doxycycline incubation caused a time-dependent increase in NURR1 mRNA (Panel A). Pre-incubation with verapamil (10 μM) abrogates KCNJ5T158A-mediated and Ang II-stimulated NURR1 mRNA and nuclear protein expression (Panel B). Expression of KCNJ5T158A and Ang II treatment stimulates the phosphorylation of ATF2 (normalized to total ATF2) (Panel C). Ca2+ channel blocker, verapamil (10 μM), inhibited the stimulatory effect of KCNJ5T158A. The western analyses for nuclear-localized NURR1 and ATF2 (Panels B and C) are representative of three independent experiments. Black and gray bars represent the absence and presence of verapamil (10 μM), respectively (Panels B and C). Results represent the mean±s.e.m. of at least five independent experiments for mRNA (Panels A and B) and three independent westerns (Panels B and C). Statistical analyses were performed using one-way ANOVA (*P<0.05 vs basal, $P<0.05 vs corresponding treatment in the absence of verapamil).

  6. Figure 6
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    Figure 6

    KCNJ5T158A activates steroidogenic acute regulatory protein (StAR) expression and post-translational modification. Cells treated with doxycycline (1 μg/mL) for 24 h, in the absence or presence of the calcium channel blocker, verapamil (10 μM), were analyzed for StAR induction by western analyses. A representative of three independent sets of protein analyses for total StAR, phosphorylated StAR and GAPDH (protein loading control) is shown in Panel A. Panels B and C represent semi-quantitative analyses by densitometry of KCNJ5T158A effects on total StAR levels and phosphorylation of StAR, respectively. Black and gray bars represent the absence and presence of verapamil (10 μM), respectively. Statistical analyses were performed using one-way ANOVA (*P<0.05 vs basal, $P<0.05 vs doxycycline treatment in the absence of verapamil).

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  1. doi: 10.1530/JME-15-0324 J Mol Endocrinol 57 1-11
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