Pharmacology and pathophysiology of mutated KCNJ5 found in adrenal aldosterone-producing adenomas.

GLUCOCORTICOIDS-CRH-ACTH-ADRENAL

Pharmacology and Pathophysiology of Mutated KCNJ5 Found in Adrenal Aldosterone-Producing Adenomas P. Tauber,* D. Penton,* J. Stindl, E. Humberg, I. Tegtmeier, C. Sterner, F. Beuschlein, M. Reincke, J. Barhanin, S. Bandulik,* and R. Warth* Medical Cell Biology (P.T., J.S., E.H., I.T., C.S., S.B., R.W.), University of Regensburg, 93053 Regensburg, Germany; Laboratoire de PhysioMédecine Moléculaire (D.P., J.B.), Centre National de la Recherche Scientifique, and Université de Nice Sophia Antipolis, FRE3472-Laboratoire de PhysioMédecine Moléculaire, 06108 Nice Cedex, France; Laboratories of Excellence, Ion Channel Science and Therapeutics (D.P., J.B.), France; and Medizinische Klinik und Poliklinik IV (F.B., M.R.), LudwigMaximilians-Universität, 80336 Munich, Germany

Somatic mutations of the potassium channel KCNJ5 are found in 40% of aldosterone producing adenomas (APAs). APA-related mutations of KCNJ5 lead to a pathological Na⫹ permeability and a rise in cytosolic Ca2⫹, the latter presumably by depolarizing the membrane and activating voltage-gated Ca2⫹ channels. The aim of this study was to further investigate the effects of mutated KCNJ5 channels on intracellular Na⫹ and Ca2⫹ homeostasis in human adrenocortical NCI-H295R cells. Expression of mutant KCNJ5 led to a 2-fold increase in intracellular Na⫹ and, in parallel, to a substantial rise in intracellular Ca2⫹. The increase in Ca2⫹ appeared to be caused by activation of voltage-gated Ca2⫹ channels and by an impairment of Ca2⫹ extrusion by Na⫹/Ca2⫹ exchangers. The mutated KCNJ5 exhibited a pharmacological profile that differed from the one of wild-type channels. Mutated KCNJ5 was less Ba2⫹ and tertiapin-Q sensitive but was inhibited by blockers of Na⫹ and Ca2⫹-transporting proteins, such as verapamil and amiloride. The clinical use of these drugs might influence aldosterone levels in APA patients with KCNJ5 mutations. This might implicate diagnostic testing of APAs and could offer new therapeutic strategies. (Endocrinology 155: 1353–1362, 2014)

ldosterone is a key regulator of salt metabolism and, thereby, influences arterial blood pressure. Primary aldosteronism is caused by bilateral adrenocortical hyperplasia or unilateral aldosterone-producing adenomas (APAs) and found in 6%–12% of patients with arterial hypertension (1). Classical physiological studies have demonstrated that control of the membrane voltage by K⫹ channels is a critical determinant of intracellular Ca2⫹ concentration and, thereby, aldosterone secretion in adrenal glomerulosa cells (for review see Ref. 2). Recently, the clinical relevance of this concept has been highlighted by establishing the link between primary aldosteronism and genetic deficits of K⫹ channel function using modern genetic approaches: mouse models for KCNK3 and KCNK9 potassium channels exhibit partial autonomy of aldosterone

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secretion (3– 6). Most notably, mutations of the KCNJ5 K⫹ channel gene have been identified as causative of familial and sporadic forms of primary aldosteronism and APAs in humans (7). Further studies have found somatic mutations of KCNJ5 in 30%– 40% of APAs (8, 9) and corroborated the clinical significance of KCNJ5 mutations. The physiological function of nonmutated KCNJ5 potassium channels and their contribution to the complex network controlling intracellular Ca2⫹ and aldosterone secretion are not fully elucidated. Wild-type KCNJ5 channels are known to be activated by G␤␥ proteins and rise in intracellular Na⫹ (10, 11). Moreover, KCNJ5 appears to modulate angiotensin II-stimulated aldosterone secretion (12). Characteristically, native glomerulosa cells show spontaneous oscillations of the membrane voltage and of the

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received October 11, 2013. Accepted January 8, 2014. First Published Online February 7, 2014

* P.T. and D.P. contributed equally to this work. S.B. and R.W. contributed equally to the work. Abbreviations: APA, aldosterone-producing adenoma; EIPA, 5-N-ethyl-N-isopropylamiloride; KB-R7943, 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate; KCN, potassium channel genes; NCKX, Na⫹/Ca2⫹/K⫹ exchanger; NCX, Na⫹/Ca2⫹ exchanger; NMDG⫹, N-methyl-D-glucamine chloride.

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Endocrinology, April 2014, 155(4):1353–1362

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Mutated KCNJ5 in Adrenal APAs

intracellular Ca2⫹ concentration (2, 13). Frequency and amplitude of these oscillations are modulated by regulators of aldosterone secretion, such as angiotensin II (6, 14). The oscillatory Ca2⫹ increases are continuously challenging intracellular Ca2⫹ homeostasis and require powerful mechanisms to prevent potentially deleterious sustained increases in cytosolic Ca2⫹. Three main mechanisms are believed to lower increased intracellular Ca2⫹ levels in glomerulosa cells: Ca2⫹ pumps localized in the membrane of the endoplasmic reticulum (sarco/endoplasmic reticulum calcium transporting ATPase), plasma membrane Ca2⫹ ATPases, and Na⫹/Ca2⫹ or Na⫹/Ca2⫹/K⫹ exchangers (members of the NCX and NCKX family) (2). For NCX-mediated Ca2⫹ export across the plasma membrane, the hyperpolarized membrane voltage and the chemical gradient of Na⫹ are required as driving force. Under conditions with depolarized membrane voltage and high intracellular Na⫹ concentration, NCX is no longer able to export Ca2⫹ but works in reverse mode and imports Ca2⫹ from the extracellular space (15). The adenoma-associated gain-of-function mutations of KCNJ5 are localized in or close to the ion selectivity filter and confer a pathological Na⫹ and Ca2⫹ permeability to the mutated channels (7, 16). According to the current concept, activity of the mutated KCNJ5 leads to influx of cations and, thereby, to membrane depolarization. The depolarization results in activation of voltagegated Ca2⫹ channels. The increased cytosolic Ca2⫹ activity ultimately promotes aldosterone secretion and adenoma formation. Because the control of intracellular Ca2⫹ is of upmost importance for the control of aldosterone secretion, it is not surprising that also mutations of a plasma membrane Ca2⫹ ATPase (ATP2B3), of the Na⫹/K⫹ ATPase (ATP1A1) (17, 18), and of a voltage-gated Ca2⫹ channel (CACNA1D) (18, 19) have been found in APAs. This study aimed at investigating the effect of adenomaassociated KCNJ5 mutations on intracellular Ca2⫹ homeostasis. Overexpression of mutant KCNJ5 in adrenocortical NCI-H295R cells increased intracellular Ca2⫹ at resting conditions and impaired Ca2⫹ export by Na⫹/ Ca2⫹ exchangers. Na⫹-permeable mutant KCNJ5 channels showed a pharmacological profile that was distinct from the one of wild-type channels. They were less sensitive to Ba2⫹ and tertiapin-Q but inhibited by blockers of Na⫹ and Ca2⫹-transporting proteins. The altered pharmacology of mutant KCNJ5 has to be considered for the interpretation of experimental and clinical data and might be relevant for the development of new therapies for APAs.

Materials and Methods Cell culture and transfection Adrenocortical carcinoma NCI-H295R cells (CLS) were cultured in a commercial cell line-specific DMEM/F12 medium con-


taining: 15mM HEPES, 6.25 ␮g/mL insulin, 6.25 ␮g/mL transferrin, 6.25 ng/mL selenium, 1.25 mg/mL bovine serum albumin, 5.35 mg/mL linoleic acid, and 2.5% Nu-Serum I (MG-42; CLS). The cells were maintained at 37°C under a humid atmosphere of 95% air/5% CO2. Plasmids (pMA-RQ [ampR]) containing the mutated cDNA sequence of human KCNJ5 (L168R, G151R, or T158A) were purchased from Invitrogen/Geneart. For expression in mammalian cells, cDNAs were subcloned into the bicistronic expression vector pIRES-CD8 (20). One day before the experiment, 1 ⫻ 106 cells were transfected with 5 ␮g of wild-type KCNJ5 or mutant KCNJ5 containing pIRES-CD8 plasmids using an electroporation system (NEON; Life Technologies GmbH). Mutant-expressing cells were always compared with wild-type KCNJ5 electroporated cells to exclude that the results are affected by electroporation-induced cellular alterations. Electroporation was done according to the manufacturer using 1-pulse 40 milliseconds at 1100 V. After electroporation, cells were cultured in antibiotic-free medium on fibronectin/collagencoated glass cover slips. The increase of KCNJ5 protein expression in transfected cells was verified by Western blotting (Supplemental Figure 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). For patch-clamp or Ca2⫹ measurements, transfected cells were identified using anti-CD8-coated dynabeads (Life Technologies GmbH).

Patch-clamp measurements Whole-cell recordings were performed at room temperature on transfected NCI-H295R cells grown on glass cover slips using an EPC-10 amplifier (Heka). Patch pipettes with a resistance of 6 –10 M⍀ were used for the recordings. The patch pipette solution contained: 95mM K-gluconate, 30mM KCl, 4.8mM Na2HPO4, 1.2mM NaH2PO4, 5mM glucose, 2.38mM MgCl2, 0.726mM CaCl2, 1mM EGTA, and 3mM ATP (pH 7.2). For some experiments, pipette solution contained 30mM NaCl instead of KCl. The extracellular Ringer-type control solution contained: 140mM NaCl, 1.8mM MgCl2, 1.8mM CaCl2, 10mM HEPES, and 5mM KCl (pH 7.4). For some experiments, bath Na⫹ was replaced by N-methyl-D-glucamine chloride (NMDG⫹) to verify the Na⫹ permeability of mutated KCNJ5 channels. For other experiments, the K⫹ concentration was increased, whereas Na⫹ was reduced to the same extent. The latter condition was to test whether mutant KCNJ5 channels transport K⫹ and Na⫹ equally well.

Ca2ⴙ measurements Intracellular Ca2⫹ levels were measured using the ratiometric fluorescent Ca2⫹ dye Fura-2 (Life Technologies GmbH). NCIH295R cells (grown on glass cover slips) were loaded at room temperature for 45 minutes with 0.5␮M Fura-2 acetoxymethyl ester in the presence of 1⫻ Power Load permeabilizing reagent (Life Technologies GmbH). The extracellular Ringer-type solution contained: 140mM NaCl, 1.8mM MgCl2, 1.8mM CaCl2, 10mM HEPES, and 5mM KCl (pH 7.4). For some experiments, bath Na⫹ was replaced by NMDG⫹. Mean fluorescence ratios of emission at 490 –530 nm after excitation at 340 and 380 nm were calculated for single-transfected cells using Axiovision software (Zeiss).


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Naⴙ and Kⴙ measurements in cell lysates NCI-H295R cells (1 ⫻ 106) were transfected with wild-type KCNJ5, mutant KCNJ5 (L168R, G151R, or T158A) containing or empty pIRES-CD8 plasmids. Transfected cells were not selected for the sodium measurement. The percentage of strongly transfected cells was usually in the range of 15%–30%. Sixteen hours after transfection, cells were quickly washed twice with Na⫹-free 300mM mannitol solution on ice. Swelling and disruption of the cells was induced by incubation in MilliQ-water on ice for 1.5 hours. Lysates were homogenated with needle and syringe (27 G) and cleared from cellular debris by centrifugation (10 min, 16 000g). Clear supernatant was used for measurements of the Na⫹ and K⫹ concentrations using flame photometry (Jenway PFP7; Bibby Scientific Ltd) with appropriate standard solutions. The ion concentrations were normalized against protein concentrations measured by a standard Bradford assay (Bio-Rad).

RNA isolation Total RNA from NCI-H295R cells, APA, and adjacent normal adrenal tissue was isolated using a column-based kit according to the manual (RNeasy Mini kit; QIAGEN). The RNA concentration was quantified with a photometer (Nanodrop; PEQLAB Biotechnologie GmbH). Quality of the RNA used for real-time RT-PCR was tested by agarose electrophoresis. The diagnosis of primary aldosteronism was established as previously reported as part of the German Conn’s Registry–Else Kröner-Fresenius Hyperaldosteronism Registry (21). Tissue sampling was approved by the local ethics committee of the University Clinic Munich and all patients provided written informed consent.

Real-time PCR Reverse transcription with Moloney Murine Leukemia Virus Reverse Transcriptase (Promega GmbH) and random primers (Fermentas GmbH) was performed using 1 ␮g total RNA. Relevant contamination with genomic DNA was excluded by negative control reactions without the reverse transcriptase enzyme. cDNA from different human tissues (human MTC panel I) was purchased from Clontech Laboratories. Real-time PCR of cDNA samples was performed on a LightCycler 480 device (Roche) using specific and intron-spanning or intron-flanking primers (Life Technologies GmbH) and a SYBR Green mastermix (Roche). Target gene expression levels in cDNA samples were quantified relative to ␤-actin expression under consideration of PCR efficiencies calculated on the basis of standard dilution curves. The specificity of PCR amplifications was verified by agarose electrophoresis and melting curve analysis. Primer sequences are listed in Supplemental Table 1.

Substances The following substances were used for the pharmacological characterization of KCNJ5: 5-N-ethyl-N-isopropyl-amiloride (EIPA), flufenamic acid, lidocaine, tertiapin-Q trifluoroacetate salt, triamterene, amiloride hydrochloride hydrate, benzamil, phenamil, bupivacain, diltiazem, nifedipine, and verapamil hydrochloride (all from Sigma-Aldrich); cariporide (Santa Cruz Biotechnology, Inc); and 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943) mesylate (Tocris Bioscience). Stock


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solutions were prepared in dimethyl sulfoxide, except of tertiapin-Q, which was dissolved in deionized water.

Statistics The numbers of experiments stated in the figures refer to the number of cells studied to calculate mean values ⫾ SEM (biological replicates). Paired or unpaired Student’s t test was used as appropriate to calculate the level of significance. Differences between groups were considered significant if P ⬍ .05.

Results Mutations of KCNJ5 lead to Naⴙ influx The adenoma-associated mutations of KCNJ5 are within or close to the selectivity filter of KCNJ5 (Figure 1D) and have in common that they make the channel permeable to other cations like Na⫹ and Ca2⫹ (7, 9, 16). To test the function of mutant KCNJ5 in an “adrenal-like” system, we expressed the KCNJ5L168R mutant and wild-type KCNJ5 in the adrenocortical carcinoma cell line NCI-H295R (22, 23). In cells transfected with wild-type KCNJ5, whole-cell currents at resting conditions were very small and not different from mock-transfected cells. After removal of Na⫹ from the bath, currents did not change, and the membrane hardly hyperpolarized (Figure 1, A and B). By contrast, the KCNJ5L168R mutant was constitutively active already at resting conditions (Figure 1A), and cells were strongly depolarized (Figure 1B). When Na⫹ was removed from the bath solution, the Na⫹ inward currents of the KCNJ5L168R mutant disappeared, and the cells hyperpolarized (Figure 1, A and B). The cytosolic Na⫹ concentration in NCI-H295R cells was at least doubled by transient transfection with mutants of KCNJ5 (L168R, G151R, and T158A) (Figure 1C). Intracellular K⫹ concentrations were slightly reduced in cells expressing mutant KCNJ5 compared with vectortransfected cells (Supplemental Figure 2) Increase of cytosolic Ca2ⴙ concentration in KCNJ5 mutant adrenal cells Next, we examined the effect of different KCNJ5 mutations on intracellular Ca2⫹ in NCI-H295R cells. Under control conditions, Ca2⫹ concentration was increased in cells expressing the mutants KCNJ5L168R, KCNJ5G151R, and KCNJ5T158A (Figure 2A). According to the present concept of the pathophysiology of mutant KCNJ5 (7, 24), increased intracellular Ca2⫹ is a consequence of the increased Na⫹ conductance, leading to depolarization and activation of voltage-gated Ca2⫹ channels. Therefore, removal of extracellular Na⫹ inducing hyperpolarization was expected to reduce intracellular Ca2⫹ in cells expressing mutant forms of KCNJ5. Surprisingly, removal of bath Na⫹ led to a strong increase of cytosolic Ca2⫹ in mutant


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Figure 1. The KCNJ5L168R mutation leads to Na⫹ influx into adrenal NCI-H295R cells. Representative whole-cell currents (A) and membrane voltage (B) of NCI-H295R cells transfected with empty vector, wild-type (WT) KCNJ5, or mutant KCNJ5L168R. Currents and voltage (n ⫽ 10 –18 per group) were measured under control (normal Ringer solution) and Na⫹-free (using NMDG⫹ instead of NaCl) conditions. Asterisk indicates P ⬍ .05 comparing control with Na⫹ free. C, Na⫹ influx leads to a substantial increase of the cytosolic Na⫹ concentration in cells with different mutants of KCNJ5 (L168R, G151R, and T158A), compared with WT KCNJ5-expressing cells; empty pIRES-CD8 vector was transfected as control (n ⫽ 3– 6 per group). Asterisks indicate P ⬍ .05 comparing empty vector or WT KCNJ5 with mutant KCNJ5. D, Scheme of KCNJ5 protein showing known mutations, all are located in or near the channel pore/selectivity filter (W126R [42], E145Q [16], E145K [18], G151R [7], G151E [16, 33], I157S [29], delI157 [43], T158A [7], and L168R [7]). The long QT syndrome type 13 (LQT13)-associated G387R mutation is located in the C terminus (44).

KCNJ5-transfected cells, whereas Ca2⫹ did not change in wild-type KCNJ5-transfected cells (Figure 2A). What is the explanation for the Ca2⫹ increase when Na⫹ is removed from the extracellular solution? The cytosolic Na⫹ concentration in mutant KCNJ5-transfected cells was increased under control conditions, compared with wild-type KCNJ5-transfected cells (Figure 1C). By removing Na⫹ from the bath solution, the concentration gradient of Na⫹ was inverted and probably provoked reverse mode of NCX in mutant KCNJ5-transfected cells. To further test the relevance of NCX reverse mode, the effect of the NCX inhibitor KB-R7943 was examined. When applied after removal of bath Na⫹, KB-R7943 (10␮M) led to a slight increase of cytosolic Ca2⫹ in wildtype cells (Figure 2B). This effect was considered nonspecific, because NCX could not export Ca2⫹ without external Na⫹, and therefore, its inhibition could not directly increase intracellular Ca2⫹. In cells expressing KCNJ5L168R, however, KB-R7943 led to a strong decrease of cytosolic

Ca2⫹ under Na⫹-free conditions, consistent with inhibition of the reverse mode of NCX (Figure 2B). In the presence of bath Na⫹, KB-R7943 led to a reduction of cytosolic Ca2⫹ concentration in cells expressing KCNJ5L168R (Figure 2C). These data suggest that the depolarization elicited by the mutant KCNJ5 was possibly sufficient to induce the reverse mode of NCX even at physiological extracellular Na⫹ concentration. In cells with wild-type KCNJ5, KB-R7943 led to an increase in cytosolic Ca2⫹ as expected for inhibition of NCX-mediated Ca2⫹ extrusion. To test the contribution of voltage-gated Ca2⫹ channels, verapamil (10␮M) was applied to transfected NCIH295R cells. In wild-type KCNJ5-transfected cells, verapamil had no effect at control conditions but inhibited the rise of Ca2⫹ caused by stimulation with high extracellular K⫹ (15mM). In cells expressing KCNJ5L168R, however, verapamil reduced the increased intracellular Ca2⫹ at control conditions to almost normal levels. High extra-


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NCX1, followed by NCX2 and NCX3. In NCI-H295R cells, NCX2 was the predominantly expressed isoform (Figure 3). All tested isoforms of Na⫹/Ca2⫹/K⫹ exchangers were expressed in adenomas and adjacent adrenal tissue. NCKX6 showed the highest expression levels in adenomas and adjacent adrenal tissues, and it was the predominantly expressed isoform in NCI-H295R cells. Among the other analyzed tissues, the brain showed the highest expression levels of NCX1, NCX2, NCKX2, and NCKX4. NCX1 was also highly expressed in the heart, whereas NCX3 and NCKX3 were enriched in the skeletal muscle. Pharmacology of mutant KCNJ5 channels The wild-type KCNJ5 is normally inactive under resting conditions and Figure 2. Fura-2 Ca2⫹ measurements of NCI-H295R cells transfected with wild-type (WT) activated by G protein-coupled recepKCNJ5 or different mutants of KCNJ5. Traces show mean values of 340 nm/380 nm ratios ⫾ tors and by high intracellular Na⫹ 2⫹ SEM as a measure of intracellular Ca concentration (WT black, L168R red, G151R blue, and concentrations (10, 11). In wild-type T158A green; n ⫽ 10 –24 per group). A, Compared with cells expressing WT KCNJ5, mutant KCNJ5-expressing cells have increased Ca2⫹ activity under control condition. Cytosolic Ca2⫹ KCNJ5-transfected NCI-H295R cells, increased at Na⫹-free conditions (Na⫹ replaced by NMDG⫹) in cells with mutant KCNJ5 but an inward current was activated by a remained stable in cells with WT KCNJ5. B, The higher [Ca2⫹]i of cells with mutant KCNJ5L168R high Na⫹ (30mM) pipette solution under Na⫹-free conditions was strongly decreased by application of KB-R7943 (10␮M). C, Effect ⫹ 2⫹ of KB-R7943 (10␮M) at physiological bath Na concentration (140mM). D, The L-type Ca in the presence of 15mM extracelluchannel blocker verapamil (10␮M) normalized Ca2⫹ activity in cells with mutant KCNJ5L168R but lar K⫹. This current was inhibited had no effect in cells with WT KCNJ5. High K⫹ (15mM) strongly increased Ca2⫹ in cells with WT when the KCNJ5 blocker tertiaKCNJ5 but had no effect in cells with mutant KCNJ5L168R. The effect of high extracellular K⫹ on intracellular Ca2⫹ was abolished by verapamil, indicating that L-type Ca2⫹ channels are activated pin-Q (25, 26) was applied to the by 15mM K⫹ in cells with WT KCNJ5. bath solution. In contrast, cells transfected with the Na⫹-permeable mutant KCNJ5L168R already showed cellular K⫹ had no clear effect, probably because cells expressing mutant KCNJ5 were already strongly depo- a huge current under resting conditions, but the current was not inhibited by tertiapin-Q (Figure 4). Similar to the larized (Figure 2D). KCNJ5G151R mutant (7), KCNJ5L168R channels were poorly blocked by Ba2⫹, a known inhibitor of wild-type Adrenal expression of Naⴙ/Ca2ⴙ and Naⴙ/Ca2ⴙ/Kⴙ KCNJ5 channels (Table 1). Conversely, KCNJ5L168R muexchanger isoforms 2⫹ The Ca measurements suggested that the reverse tant channels were inhibited by various blockers of Na⫹ mode of NCX contributed to the increased Ca2⫹ in cells channels, Ca2⫹ channels, and Na⫹/Ca2⫹ exchangers (Figexpressing KCNJ5L168R. Na⫹/Ca2⫹/K⫹ exchangers hardly ure 5 and Table 1). EIPA, a blocker of Na⫹/H⫹ exchangwork in reverse mode and extrude Ca2⫹ out of the cells. To ers, was potently blocking KCNJ5L168R (IC50 ⫽ 0.6␮M) test which isoforms of the Na⫹/Ca2⫹ and Na⫹/Ca2⫹/K⫹ (Figure 5A), whereas amiloride blocked less potently (IC50 exchanger family might be relevant for the phenotype, the ⫽ 12␮M) (Figure 5D). The NCX inhibitor KB-R7943 was mRNA expression levels in human APAs, adjacent adrenal potently blocking KCNJ5L168R with IC50 ⫽ 0.8␮M, tissue, and NCI-H295R cells were quantified by real-time which was lower than the reported IC50 for NCX (Figure PCR and compared with the expression in other normal 5B) (27). Among the tested blockers of L-type voltagehuman tissues. In adenomas and adjacent adrenal tissue, the gated Ca2⫹ channels, verapamil strongly inhibited most abundant isoform of Na⫹/Ca2⫹ exchangers was KCNJ5L168R mutant channels (IC50 ⫽ 1.2␮M), followed


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Discussion The discovery that somatic mutations of KCNJ5 confer a pathological Na⫹ permeability to the mutated channel pointed to disturbed ion homeostasis and membrane voltage control as determinants of the pathogenesis of APAs (9, 19). Here, we show that mutant KCNJ5 led to depolarization and increased intracellular Na⫹ and Ca2⫹ concentrations in the adrenocortical cell line NCI-H295R. High intracellular Na⫹ impaired Ca2⫹ export via Na⫹/ Ca2⫹ exchangers (NCX) and possibly allowed Ca2⫹ influx through NCX working in reverse transport mode. Interestingly, Ca2⫹ channel blockers, such as verapamil, and Na⫹ channel blockers, such as amiloride, inhibited the mutated KCNJ5 channel. Mutations of KCNJ5 lead to Naⴙ influx and intracellular Naⴙ accumulation Sequencing of KCNJ5 from APAs revealed a number of mutations, which all interfere with the ion selectivity filter, leading to Na⫹ influx into the cells (7, 16, 28 –30). According to the present concept, Na⫹ influx results in membrane depolarization, activation of voltage-dependent Ca2⫹ channels, and finally stimulation of steroid hormone synthesis and cell proliferation. However, experimental data about the cellular consequences of mutations of by diltiazem (IC50 ⫽ 11␮M) (Figure 5E). Nifedipine was KCNJ5 are sparse, and most of the electrophysiological weak inhibitor of the KCNJ5L168R mutant (IC50 ⫽ 53␮M) characterization of KCNJ5 mutants has been performed in (Figure 5F). Representative traces of the effect of potential heterologous expression systems (7, 16, 28, 29, 31–33). inhibitors of the mutant KCNJ5L168R are show in SuppleHere, we used NCI-H295R cells electroporated with mental Figure 3. Verapamil also blocked the G151R mumutant KCNJ5 channels to study the pathophysiology of tant of KCNJ5 (69 ⫾ 12% remaining Na⫹ inward current these channels in an adrenocortical cell model. In native at 1␮M verapamil and 43 ⫾ 9% at 10␮M, n ⫽ 13) and the tissues, such as the heart, KCNJ5 forms heteromeric K⫹ T158A mutant (87 ⫾ 12% remaining Na⫹ inward current channels with KCNJ3 (34). Because adrenal cells express at 1␮M verapamil and 50 ⫾ 14% at 10␮M, n ⫽ 8). several members of the KCNJ family (7), we expressed KCNJ5 without KCNJ3 assuming that exogenous KCNJ5 associates with its physiological partner in adrenocortical cells. Under our experimental conditions, wild-type KCNJ5 was hardly active but appeared to be enhanced by increased cytosolic Na⫹ concentration (Figure 4). However, cells with the mutant KCNJ5L168R were strongly depolarized under control conditions and showed large inward currents that Figure 4. The L168R mutation of KCNJ5 abrogates inhibition by tertiapin-Q (TQ). NCI-H295R disappeared under Na⫹-free condicells were transfected with WT KCNJ5 or mutant KCNJ5L168R. Whole-cell currents were measured tions. Mutations of KCNJ5 could by patch-clamp under conditions, where WT KCNJ5 current was detectable (pipette solution with 30mM Na⫹ to activate WT KCNJ5; bath solution with 15mM K⫹ to increase the inward current have led to G protein-independent of K⫹). A, Representative traces show that the KCNJ5 blocker TQ (1␮M) inhibited the inward activation of the channel, similar to current in cells with WT KCNJ5 but not with KCNJ5L168R. B, Mean values of the TQ sensitive the situation of the “weaver mutant” current at different voltage clamp steps show the inward rectification of WT KCNJ5 channels of KCNJ6, which is characterized by (n ⫽ 12), whereas the TQ sensitivity was lost in mutant KCNJ5L168R channels (n ⫽ 6).

Figure 3. mRNA expression of Ca2⫹ transporters. mRNA expression of different Na⫹/Ca2⫹ exchangers and Na⫹/K⫹/Ca2⫹ exchangers in NCIH295R cells (n ⫽ 4), normal adjacent adrenal gland (n ⫽ 3), APA (n ⫽ 3), and different human tissues (n ⫽ 1 per tissue, pooled samples from a commercial cDNA panel). Gene expression was quantified by realtime PCR and normalized to ␤-actin expression. Mean values were calculated for NCI-H295R cells and adrenal tissue cDNA. Relative expression levels of each transcript compared with the expression in brain are shown in a color scale (green color, minimum expression level; red, maximum expression level; yellow, median, color range is sorted by the rank of the log2 relative expression levels).


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Table 1.


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Pharmacological Profile of the Mutant KCNJ5L168R

Remaining current at ⴚ120 Substance Concentration mV (% of control ⴞ SEM) n

Classical targets (inhibitors of) Pharmacological group

EIPA

10␮M

15.4 ⫾ 3.7

Benzamil

10␮M

22.2 ⫾ 4.6

Phenamil

10␮M

27.5 ⫾ 5.8

13 Na⫹/H⫹ exchanger, polycystic kidney disease 2-like 1 (TRPP3) 11 Epithelial sodium channel alpha subunit (ENaC) and Na⫹/Ca2⫹ exchanger 8 ENaC

Amilorid Triamteren Bupivacain Lidocain KB-R7943

10␮M 10␮M 10␮M 100␮M 10␮M

55.0 ⫾ 1.6 85.4 ⫾ 5.2 16.6 ⫾ 7.0 66.4 ⫾ 8.3 5.8 ⫾ 1.5

9 6 5 6 9

Verapamil Diltiazem Nifedipine Cariporide Flufenamate

10␮M 10␮M 10␮M 100␮M 100␮M

20.5 ⫾ 5.6 53.9 ⫾ 3.1 87.2 ⫾ 6.6 65.9 ⫾ 6.7 88.9 ⫾ 2.6

9 6 6 8 7

74.4 ⫾ 2.9 103.6 ⫾ 11.8

5 6

Ba2⫹ 5mM Tertiapin-Q 1␮M

ENaC and T-type Ca2⫹ channels ENaC Voltage-gated Na⫹ channels Voltage-gated Na⫹ channels Na⫹/Ca2⫹ exchanger, ion channels L-type Ca2⫹ channels L-type Ca2⫹ channels L-type Ca2⫹ channels Na⫹/H⫹ exchanger Cyclooxygenase, hyaluronidase, unspecific cation channels K⫹ channels K⫹ channels (KCNJ5, KCNJ3, KCNJ1, KCNMA1)

Analogue of amiloride Analogue of amiloride/ diuretic Analogue of amiloride/ diuretic Diuretic Pteridine/diuretic Local anesthetic Local anesthetic Phenylalkylamine Benzothiazepine Dihydropyridine

Toxin from honey bee

The mutant KCNJ5L168R was overexpressed in NCI-H295R cells. The resulting Na⫹-depending inward current was measured at ⫺120 mV. Data are shown as % of the remaining current after treatment of the cells with the respective substance compared with the current under control solution (composition of the solutions is given in Materials and Methods). Experiments with Ba2⫹ and tertiapin-Q were done using different solutions in order to activate wild-type KCNJ5 (Ba2⫹ with 50mM K⫹ in bath solution, tertiapin-Q with 15mM K⫹ in the bath, and 30mM NaCl instead of KCl in the pipette solution).

a loss of ion selectivity and G␤␥ insensitivity (35). Probably, Na⫹ influx through mutated KCNJ5 leads, via an increase of the cytosolic Na⫹ concentration, to Na⫹-dependent activation of KCNJ5, a deleterious positive feedback aggravating the severity of the mutation. Oki et al (24) reported a mild increase of the cytosolic Na⫹ concentration in HAC15 cells expressing mutated KCNJ5T158A using a Na⫹-sensitive fluorescent dye. Using flame photometry, we found a substantial increase (2fold) of cytosolic Na⫹ concentration in lysates of NCIH295R cells expressing mutated KCNJ5L168R. Future studies are required to test whether the Na⫹ content of adenomas with KCNJ5 mutations is higher compared with adenomas without KCNJ5 mutations or adjacent normal adrenal tissue. Increase of cytosolic Ca2ⴙ concentration in KCNJ5 mutant adrenal cells The increase of cytosolic Ca2⫹ is considered a critical event in the pathogenesis of APAs containing mutated KCNJ5. In fact, Ca2⫹ was found increased in HAC15 cells expressing mutant KCNJ5T158A (24) and in HEK293T cells expressing mutant KCNJ5G151E or KCNJ5Y152C (32). This concept has been further corroborated by the discovery of APA-associated gain-of-function mutations of the L-type Ca2⫹ channel CACNA1D (18, 19). Here, we

provide direct experimental evidence that the most frequent KCNJ5 mutations found in APAs (KCNJ5G151R and KCNJ5L168R) lead to an increase of cytosolic Ca2⫹ in adrenal cells. Cytosolic Ca2⫹ activity is the sum of Ca2⫹ influx and efflux across the plasma membrane and of Ca2⫹ movements between the cytosol and intracellular stores. The pathological Na⫹ conductance of mutated KCNJ5 depolarizes the membrane and activates voltage-gated Ca2⫹ channels (7). The L-type Ca2⫹ channel blocker nifedipine reduces aldosterone synthase expression and aldosterone levels in cells expressing mutant KCNJ5 (24, 32). These data suggest that membrane depolarization, leading to activation of voltage-gated Ca2⫹ channels, is the critical event, leading to the rise of cytosolic Ca2⫹, aldosterone secretion, and adenoma formation (7). Therefore, we expected a decrease of internal Ca2⫹ when mutated KCNJ5-expressing cells hyperpolarized upon removal of extracellular Na⫹. Surprisingly, the opposite was observed, and despite the hyperpolarization (Figure 1B), intracellular Ca2⫹ activity increased (Figure 2A). The high intracellular Na⫹ concentration of mutated KCNJ5-expressing cells suggested that influx through Na⫹/Ca2⫹ exchangers (NCX) working in reverse mode could contribute to the increase in Ca2⫹, as described for cardiac myocytes (36).


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expansion of mutant cells and APA formation (7). Such a mechanism has also been described in gastric myofibroblasts and pulmonary artery smooth muscle cells, where proliferation was accelerated by reverse mode of NCX (37, 38). On the other hand, for a positive effect on proliferation, Ca2⫹ should not exceed a critical level. Overwhelming increases of cytosolic Ca2⫹ appear to induce apoptosis rather than increased proliferation as evidenced by KCNJ5 mutants that exhibit very high Na⫹ permeability.

Figure 5. Pharmacology of mutant KCNJ5L168R channels. NCI-H295R cells were transfected with mutant KCNJ5L168R, and inward currents at ⫺120 mV were measured under control conditions and after application of increasing concentrations of blockers. Mean values ⫾ SEM of the remaining current (% of control) after treatment with the blocker are shown (n ⫽ 4 –15), and fitted curves were used to determine IC50 values. The results of additional blockers are shown in Table 1.

Is it possible that the high intracellular Na⫹ concentration allows reverse mode of NCX even under control conditions? The direction of NCX-mediated transport is dependent on 2 parameters, the membrane voltage (hyperpolarization supports Ca2⫹ extrusion/Na⫹ import; depolarization favors Ca2⫹ import/Na⫹ export) and the concentration gradients for Ca2⫹ and Na⫹ (Supplemental Figure 4). Although the Ca2⫹-lowering effect of KBR7943 is not a prove for the reverse mode of NCX, the depolarization and the high intracellular Na⫹ make it very likely that NCX operates in reverse mode in adenoma cells with strong expression of NCX (Figure 3) and KCNJ5 mutations. The increased cytosolic Ca2⫹ is supposed to stimulate proliferation and, thereby, to promote clonal

Pharmacology of mutant KCNJ5 channels The APA-associated mutations of KCNJ5 profoundly modified pharmacology of the channel. Tertiapin-Q, a known KCNJ5 inhibitor (25, 26), is only a weak inhibitor of mutated KCNJ5 (Table 1) (28). The NCX blocker KBR7943, however, was a potent inhibitor of KCNJ5L168R (Table 1), but it probably also blocks wild-type KCNJ5 (39). KCNJ5L168R was inhibited by the K⫹-sparing diuretic amiloride (IC50 ⫽ 12␮M) and, more potently, by the L-type Ca2⫹ channel blocker verapamil (IC50 ⫽ 1.2␮M). The G151R and T158A mutants of KCNJ5 were also blocked by verapamil but less potently. At high therapeutic doses of verapamil (3 ⫻ 120 mg/d), plasma concentrations are in the range of 1–2 ␮mol/L (40), sufficient to block the L168R mutant. Probably, verapamil has a dual effect. It protects against the negative effects of mutated KCNJ5 by directly blocking the mutated channel and by inhibiting depolarization-activated voltage-gated Ca2⫹ channels. As a consequence, aldosterone secretion and clonal proliferation might be reduced. This could have 2 important clinical implications for APA patients with KCNJ5 mutations. 1) Although verapamil is considered to have minimal effects on plasma aldosterone (41), administration before diagnostic blood sampling could influence plasma aldosterone levels and could lead to false low aldosterone/renin ratio, especially in patients with KCNJ5 mutations. 2) High therapeutic doses of verapamil and more potent inhibitors of mutated KCNJ5 (alone or in combination with amiloride) could be useful to improve hyperaldosteronism and hypokalemia in patients with KCNJ5 mutations. Maybe, such substances are also capable to reduce the deleterious effects of mutated KCNJ5 on cell proliferation and promote regression of existing adenomas.

Acknowledgments Address all correspondence and requests for reprints to: Dr Sascha Bandulik, Medical Cell Biology, University of Regens-


doi: 10.1210/en.2013-1944

burg, Universitaetsstrasse 31, 93053 Regensburg, Germany. Email: [email protected]. This work was supported by the Deutsche Forschungsgemeinschaft Grants FOR1086 (to R.W. and S.B.) and Re 752/ 17–1 (to F.B. and M.R.) and by the European Section of Aldosterone Council (J.B.), a French Agence Nationale pour la Recherche (ANR) BeyondTASKs grant (J.B.), the LabEx Ion channel Science and Therapeutics Grant ANR-11-LABX0015– 01 (to J.B.), the Centre national de la recherche scientifique (CNRS) Project for International Scientific Cooperation (J.B.), and the Else Kröner-Fresenius Foundation, Germany (M.R.). Disclosure Summary: The authors have nothing to disclose.

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