The physiological control of steroid hormone secretion from the adrenal cortex depends on the function of potassium channels. The "two-pore domain K(+) channels" (K2P) TWIK-related acid sensitive K(+) channel 1 (TASK1), TASK3, and TWIK-related K(+) channel 1 (TREK1) are strongly expressed in adrenocortical cells. They confer a background K(+) conductance to these cells which is important for the K(+) sensitivity as well as for angiotensin II and adrenocorticotropic hormone-dependent stimulation of aldosterone and cortisol synthesis. Mice with single deletions of the Task1 or Task3 gene as well as Task1/Task3 double knockout mice display partially autonomous aldosterone synthesis. It appears that TASK1 and TASK3 serve different functions: TASK1 affects cell differentiation and prevents expression of aldosterone synthase in the zona fasciculata, while TASK3 controls aldosterone secretion in glomerulosa cells. TREK1 is involved in the regulation of cortisol secretion in fasciculata cells. These data suggest that a disturbed function of K2P channels could contribute to adrenocortical pathologies in humans.
Understanding the function of the KCNJ5 potassium channel through characterization of naturally occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism.
Elucidation of the molecular mechanisms leading to autonomous aldosterone secretion is a prerequisite to define potential targets and biomarkers in the context of primary aldosteronism. After a genome-wide association study with subjects from the population-based Cooperative Health Research in the Region of Augsburg F4 survey, we observed a highly significant association (P=6.78×10(-11)) between the aldosterone to renin ratio and a locus at 5q32. Hypothesizing that this locus may contain genes of relevance for the pathogenesis of primary aldosteronism, we investigated solute carrier family 26 member 2 (SLC26A2), a protein with known transport activity for sulfate and other cations. Within murine tissues, adrenal glands showed the highest expression levels for SLC26A2, which was significantly downregulated on in vivo stimulation with angiotensin II and potassium. SLC26A2 expression was found to be significantly lower in aldosterone-producing adenomas in comparison with normal adrenal glands. In adrenocortical NCI-H295R cells, specific knockdown of SLC26A2 resulted in a highly significant increase in aldosterone secretion. Concomitantly, expression of steroidogenic enzymes, as well as upstream effectors including transcription factors such as NR4A1, CAMK1, and intracellular Ca(2+) content, was upregulated in knockdown cells. To substantiate further these findings in an SLC26A2 mutant mouse model, aldosterone output proved to be increased in a sex-specific manner. In summary, these findings point toward a possible effect of SLC26A2 in the regulation of aldosterone secretion potentially involved in the pathogenesis of primary aldosteronism.
In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown.
Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein-activated inward rectifier K(+) channel 4, GIRK4, account for ?40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na(+)/K(+)-ATPase 1 and Ca(2+)-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na(+)/K(+)-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na(+) activation of phosphorylation and K(+) inhibition of phosphorylation that indicate decreased Na(+) and K(+) binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na(+)/K(+)-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.
Primary aldosteronism is a heterogeneous group of disorders comprising both sporadic and familial forms. Mutations in the KCNJ5 gene, which encodes the inward rectifier K(+) channel 4 (G protein-activated inward rectifier K(+) channel 4, Kir3.4), cause familial hyperaldosteronism type III (FH-III) and are involved in the pathogenesis of sporadic aldosterone-producing adenomas.
Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.
Task3 K(+) channels are highly expressed in the adrenal cortex and contribute to the angiotensin II and K(+) sensitivity of aldosterone-producing glomerulosa cells. Adult Task3(-/-) mice display a partially autonomous aldosterone secretion, subclinical hyperaldosteronism, and salt-sensitive hypertension. Here, we investigated the age dependence of the adrenal phenotype of Task3(-/-) mice. Compared with adults, newborn Task3(-/-) mice displayed a severe adrenal phenotype with strongly increased plasma levels of aldosterone, corticosterone, and progesterone. This adrenocortical dysfunction was accompanied by a modified gene expression profile. The most strongly up-regulated gene was the protease renin. Real-time PCR corroborated the strong increase in adrenal renin expression, and immunofluorescence revealed renin-expressing cells in the zona fasciculata. Together with additional factors, activation of the local adrenal renin system is probably causative for the severely disturbed steroid hormone secretion of neonatal Task3(-/-) mice. The changes in gene expression patterns of neonatal Task3(-/-) mice could also be relevant for other forms of hyperaldosteronism.
Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na(+)/K(+) ATPase ? subunit) and ATP2B3 (encoding a Ca(2+) ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.
Primary aldosteronism is the most frequent cause of endocrine hypertension. Three forms of familial hyperaldosteronism (FH) have been described, named FH-I to -III. Recently, a mutation of KCNJ5 has been shown to be associated with FH-III, whereas the cause of FH-II is still unknown. In this study we searched for mutations in KCNJ5 in 46 patients from 21 families with FH, in which FH-I was excluded. We identified a new germline G151E mutation in 2 primary aldosteronism-affected subjects from an Italian family and 3 somatic mutations in aldosterone-producing adenomas, T158A described previously as a germline mutation associated with FH-III, and G151R and L168R both described as somatic mutations in aldosterone-producing adenoma. The phenotype of the family with the G151E mutation was remarkably milder compared with the previously described American family, in terms of both clinical and biochemical parameters. Furthermore, patients with somatic KCNJ5 mutations displayed a phenotype indistinguishable from that of sporadic primary aldosteronism. The functional characterization of the effects of the G151E mutation in vitro showed a profound alteration of the channel function, with loss of K(+) selectivity, Na(+) influx, and membrane depolarization. These alterations have been postulated to be responsible for voltage gate Ca(2+) channel activation, increase in cytosolic calcium, and stimulation of aldosterone production and adrenal cell proliferation. In conclusion, we describe herein a new mutation in the KCNJ5 potassium channel associated with FH-III, responsible for marked alterations of channel function but associated with a mild clinical and hormonal phenotype.
Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion (J(K)) and Na absorption (J(Na)) at slow (?1) and fast (?5 nl·min(-1)·mm(-1)) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal J(K), J(Na), or the flow-induced [Ca(2+)](i) transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce J(K). Basolateral Cl removal reversibly inhibited FIKS but not basal J(K) or J(Na). Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH(4)(+) at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH(4)(+) addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH(4)(+) entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.
The K+ channel expressed by the KCNJ10 gene (Kir4.1) has previously demonstrated importance in retinal function in animal experiments. Recently, mutations in KCNJ10 were recognised as pathogenic in man, causing a constellation of symptoms, including epilepsy, ataxia, sensorineural deafness and a renal tubulopathy designated as EAST syndrome. We have studied the impact of KCNJ10 mutations on the human electroretinogram (ERG) in four unrelated patients with EAST syndrome. Corneal ganzfeld ERGs were elicited in response to flash stimuli of strengths of 0.001–10 phot cd s/m2 presented scotopically, and 0.3–10 phot cd s/m2 presented photopically. ERG waveforms from light-adapted retinae of all patients showed reduced amplitudes of the photopic negative response (PhNR) (P < 0.001). The photopic ERGs showed a delay in b-wave time to peak, but the photopic hill, i.e. the relative variation of time to peak and amplitude with luminance flash strength, was preserved. Scotopic ERGs to flash strengths 0.01 to 0.1 phot cd s/m2 showed a delay of up to 20 ms before the onset of the b-wave in two patients compared to controls. Stimulus–response functions were fitted by Michaelis–Menten equations and showed significantly lower retinal sensitivity in two patients than in controls (P < 0.001). Our study for the first time in the human ERG shows changes in association with KCNJ10 mutations affecting a Muller cell K+ channel. These data illustrate the role of KCNJ10 function in the physiology of proximal and possibly also the distal human retina.
Mutations in the K+ channel gene KCNJ10 (Kir4.1) cause the autosomal recessive EAST syndrome which is characterized by epilepsy, ataxia, sensorineural deafness, and a salt-wasting tubulopathy. The renal salt-wasting pathology of EAST syndrome is caused by transport defects in the distal convoluted tubule where KCNJ10 plays a pivotal role as a basolateral K+ channel. This review on EAST syndrome outlines the molecular aspects of the physiology and pathophysiology of KCNJ10 in the distal convoluted tubule.
Mutations of the KCNJ10 (Kir4.1) K(+) channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henles loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.
The general transcription factor II H (TFIIH), comprised of a core complex and an associated CAK-complex, functions in transcription, DNA repair and cell cycle control. Mutations of the two largest subunits, p89 (XPB) and p80 (XPD), cause the hereditary cancer-prone syndrome xeroderma pigmentosum.
Task2 K(+) channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2(-/-) mice showed disturbed chemosensory function with hypersensitivity to low CO(2) concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2(-/-) mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem-spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O(2) chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.
The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs. Mice lacking ANO1 expression exhibit transport defects and a pathology similar to cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Here we analyzed expression of all ten members (ANO1-ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane but only ANO1, 2, 6, and 7 produced Ca(2+)-activated Cl(-) conductance, as analyzed by ATP-induced iodide quenching of YFP fluorescence. In contrast ANO9 and ANO10 suppressed baseline Cl(-) conductance and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1 also ANO6 and 10 produced chloride currents, albeit with very different Ca(2+) sensitivity and activation time. We conclude that each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca(2+)-dependent Cl(-) channels.
The KCNE3 beta-subunit constitutively opens outwardly rectifying KCNQ1 (Kv7.1) K(+) channels by abolishing their voltage-dependent gating. The resulting KCNQ1/KCNE3 heteromers display enhanced sensitivity to K(+) channel inhibitors like chromanol 293B. KCNE3 was also suggested to modify biophysical properties of several other K(+) channels, and a mutation in KCNE3 was proposed to underlie forms of human periodic paralysis. To investigate physiological roles of KCNE3, we now disrupted its gene in mice. kcne3(-/-) mice were viable and fertile and displayed neither periodic paralysis nor other obvious skeletal muscle abnormalities. KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they might facilitate transepithelial Cl(-) secretion through basolateral recycling of K(+) ions and by increasing the electrochemical driving force for apical Cl(-) exit. Indeed, cAMP-stimulated electrogenic Cl(-) secretion across tracheal and intestinal epithelia was drastically reduced in kcne3(-/-) mice. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3(-/-) mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. Further, these results suggest KCNE3 as a potential modifier gene in cystic fibrosis.
Tandem of P domains in a weak inwardly rectifying K(+) channel 1 (TWIK1) is a K(+) channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT(1)R or adrenoreceptor alpha2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G(i) protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.
In the past, a number of candidates have been proposed to form Ca(2+) activated Cl(-) currents, but it is only recently that two families of proteins, the bestrophins and the TMEM16-proteins, recapitulate reliably the properties of Ca(2+) activated Cl(-) currents. Bestrophin 1 is strongly expressed in the retinal pigment epithelium, but also at lower levels in other cell types. Bestrophin 1 may form Ca(2+) activated chloride channels and, at the same time, affect intracellular Ca(2+) signaling. In epithelial cells, bestrophin 1 probably controls receptor mediated Ca(2+) signaling. It may do so by facilitating Ca(2+) release from the endoplasmic reticulum, thereby indirectly activating membrane localized Ca(2+)-dependent Cl(-) channels. In contrast to bestrophin 1, the Ca(2+) activated Cl(-) channel TMEM16A (anoctamin 1, ANO1) shows most of the biophysical and pharmacological properties that have been attributed to Ca(2+)-dependent Cl(-) channels in various tissues. TMEM16A is broadly expressed in both mouse and human tissues and is of particular importance in epithelial cells. Thus exocrine gland secretion as well as electrolyte transport by both respiratory and intestinal epithelia requires TMEM16A. Because of its role for Ca(2+)-dependent Cl(-) secretion in human airways, it is likely to become a prime target for the therapy of cystic fibrosis lung disease, caused by defective cAMP-dependent Cl(-) secretion. It will be very exciting to learn, how TMEM16A and other TMEM16-proteins are activated upon increase in intracellular Ca(2+), and whether the other nine members of the TMEM16 family also form Cl(-) channels with properties similar to TMEM16A.
Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy).
The positively charged fluorescent dyes ethidium (Et(+)) and propidium (Pr(2+)) are widely used as DNA and necrosis markers. Et(+) is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (< or =500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et(+) accumulated predominantly in the S2/S3 segments of the PT, but not Pr(2+). In cells stably overexpressing human OCTs (hOCTs), Et(+) uptake was observed with K(m) values of 0.8 +/- 0.2 microM (hOCT1), 1.7 +/- 0.5 microM (hOCT2), and 2.0 +/- 0.5 microM (hOCT3), whereas Pr(2+) was not transported. Accumulation of Et(+) was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP(+)), cimetidine, and tetraethylammonium (TEA(+)). For hOCT1 and hOCT2, the IC(50) values for MPP(+), TEA(+), and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et(+) uptake by MPP(+) and cimetidine was shown to be competitive. Et(+) also inhibited transport of 0.1 microM [(3)H]MPP(+) by all hOCT isoforms with IC(50) values between 0.4 and 1.3 microM, and the inhibition of hOCT1-mediated uptake of MPP(+) by Et(+) was competitive. In Oct1/2(-/-) mice, Et(+) uptake in the PT was almost abolished. The data demonstrate that Et(+) is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et(+) and their strong expression in various organs, strict safety guidelines for Et(+) handling should be reinforced.
Primary aldosteronism (PA, autonomous aldosterone production from the adrenal cortex) causes the most common form of secondary arterial hypertension (HT), which is also the most common curable form of HT. Recent studies have highlighted an important role of mutations in genes encoding potassium channels in the pathogenesis of PA, both in human disease and in animal models. Here, we have exploited the unique features of the hyperaldosteronemic phenotype of Kcnk3 null mice, which is dependent on sexual hormones, to identify genes whose expression is modulated in the adrenal gland according to the dynamic hyperaldosteronemic phenotype of those animals. Genetic inactivation of one of the genes identified by our strategy, dickkopf-3 (Dkk3), whose expression is increased by calcium influx into adrenocortical cells, in the Kcnk3 null background results in the extension of the low-renin, potassium-rich diet insensitive hyperaldosteronemic phenotype to the male sex. Compound Kcnk3/Dkk3 animals display an increased expression of Cyp11b2, the rate-limiting enzyme for aldosterone biosynthesis in the adrenal zona glomerulosa (ZG). Our data show that Dkk3 can act as a modifier gene in a mouse model for altered potassium channel function and suggest its potential involvement in human PA syndromes.
Task1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K(+) channel-related acid-sensitive K(+) channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3(-/-) mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3(-/-) mice were strongly depolarized compared with wild-type (-52 vs. -79 mV), and in fresh adrenal slices Ca(2+) signaling of Task3(-/-) glomerulosa cells was abnormal. In living Task3(-/-) mice, the regulation of aldosterone secretion showed specific deficits: Under low Na(+) and high K(+) diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na(+) and low K(+) diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3(-/-) mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na(+) diets. Isolated adrenal glands of Task3(-/-) produced 2-fold more aldosterone. As a consequence, Task3(-/-) mice showed salt-sensitive arterial hypertension (plus 10 mm Hg). In conclusion, Task3 plays an important role in the adaptation of aldosterone secretion to dietary salt intake.
TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic ? cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.
Visinin-like 1 (VSNL1) is upregulated in aldosterone-producing adenomas (APAs) compared with normal adrenals. We demonstrate that VSNL1 overexpression in adrenocortical carcinoma cells (NCI H295R) upregulates basal and angiotensin II-stimulated CYP11B2 gene expression 3.2- and 1.5-fold, respectively. Conversely, silencing VSNL1 by RNA interference decreases angiotensin II-stimulated CYP11B2 expression and aldosterone secretion by 41.0% and 34.5%, respectively. Mutations in the potassium channel KCNJ5 have been identified in APAs that result in sodium influx and membrane depolarization and are postulated to result in calcium influx in adrenal glomerulosa cells. VSNL1 and CYP11B2 are 8.1- and 6.0-fold more highly expressed, respectively, in APAs harboring KCNJ5 mutations compared with those without, and the upregulation of VSNL1 in these APAs accounts for the overexpression of VSNL1 in the total APA sample set compared with normal adrenals. Silencing VSNL1 in H295R cells renders them sensitive to ionomycin-induced apoptosis, indicating that VSNL1 protects these cells against calcium-induced cell death. Concomitant expression of mutated KCNJ5 (G151R) and silencing VSNL1 results in apoptosis of H295R cells, an effect that is blocked by nifedipine and is absent using a control small-interfering RNA or when wild-type KCNJ5 is expressed and VSNL1 is silenced. These data demonstrate that VSNL1 plays a dual function in vitro in the regulation of CYP11B2 gene expression and in the inhibition of calcium-induced apoptosis. In addition, VSNL1 may play a role in the pathophysiology of APAs harboring mutations in the potassium channel KCNJ5 via its antiapoptotic function in response to calcium cytotoxicity and its effect on aldosterone production.
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