The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis.

Misalignment of physiological circadian rhythms promotes obesity which is characterized by white adipose tissue (WAT) expansion. Differentiation of Adipose stem/progenitor cells (ASCs) contributes to WAT increase but the importance of the cellular clock in this process is incompletely understood. In the present study, we reveal the role of the circadian transcription factor Aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) in human ASCs, isolated from subcutaneous (s)WAT samples of patients undergoing routine elective plastic abdominal surgery. We show that circadian synchronization by serum-shock or stimulation with adipogenic stimuli leads to a different expression pattern of ARNTL2 relative to its well-studied paralogue ARNTL1. We demonstrate that ARNTL2 mRNA is downregulated in ASCs upon weight-loss (WL) whereas ARNTL2 protein is rapidly induced in the course of adipogenic differentiation and highly abundant in adipocytes. ARNTL2 protein is maintained in ASCs cooperatively by mechanistic Target of Rapamycin (mTOR) and Mitogen-activated Protein Kinase (MAPK) signalling pathways while ARNTL2 functions as an inhibitor on both circuits, leading to a feedback mechanism. Consistently, ectopic overexpression of ARNTL2 repressed adipogenesis by facilitating the degradation of ARNTL1, inhibition of Kruppel-Like Factor 15 (KLF15) gene expression and down-regulation of the MAPK-CCAAT/enhancer-binding protein ß (C/EBPß) axis. Western blot analysis of sWAT samples from normal-weight, obese and WL donors revealed that ARNTL2 protein was solely elevated by WL compared to ARNTL1 which underscores unique functions of both transcription factors. In conclusion, our study reveals ARNTL2 to be a WL-regulated inhibitor of adipogenesis which might provide opportunities to develop strategies to ameliorate obesity.

Dipeptidyl peptidase-4 cell surface expression marks an abundant adipose stem/progenitor cell population with high stemness in human white adipose tissue.

The capacity of adipose stem/progenitor cells (ASCs) to undergo self-renewal and differentiation is crucial for adipose tissue homoeostasis, regeneration and expansion. However, the heterogeneous ASC populations of the adipose lineage constituting adipose tissue are not precisely known. In the present study, we demonstrate that cell surface expression of dipeptidyl peptidase-4 (DPP4)/cluster of differentiation 26 (CD26) subdivides the DLK1-/CD34+/CD45-/CD31- ASC pool of human white adipose tissues (WATs) into two large populations. Ex vivo, DPP4+ ASCs possess higher self-renewal and proliferation capacity and lesser adipocyte differentiation potential than DDP4- ASCs. The knock-down of DPP4 in ASC leads to significantly reduced proliferation and self-renewal capacity, while adipogenic differentiation is increased. Ectopic overexpression of DPP4 strongly inhibits adipogenesis. Moreover, in whole mount stainings of human subcutaneous (s)WAT, we detect DPP4 in CD34+ ASC located in the vascular stroma surrounding small blood vessels and in mature adipocytes. We conclude that DPP4 is a functional marker for an abundant ASC population in human WAT with high proliferation and self-renewal potential and low adipogenic differentiation capacity.

An organoid model derived from human adipose stem/progenitor cells to study adipose tissue physiology.

We established a functional adipose organoid model system for human adipose stem/progenitor cells (ASCs) isolated from white adipose tissue (WAT). ASCs were forced to self-aggregate by a hanging-drop technique. Afterwards, spheroids were transferred into agar-coated cell culture dishes to avoid plastic-adherence and dis-aggregation. Adipocyte differentiation was induced by an adipogenic hormone cocktail. Morphometric analysis revealed a significant increase in organoid size in the course of adipogenesis until d 18. Whole mount staining of organoids using specific lipophilic dyes showed large multi- and unilocular fat deposits in differentiated cells indicating highly efficient differentiation of ASCs into mature adipocytes. Moreover, we found a strong induction of the expression of key adipogenesis and adipocyte markers (CCAAT/enhancer-binding protein (C/EBP) ß, peroxisome proliferator-activated receptor (PPAR) γ, fatty acid-binding protein 4 (FABP4), adiponectin) during adipose organoid formation. Secreted adiponectin was detected in the cell culture supernatant, underscoring the physiological relevance of mature adipocytes in the organoid model. Moreover, colony formation assays of collagenase-digested organoids revealed the maintenance of a significant fraction of ASCs within newly formed organoids. In conclusion, we provide a reliable and highly efficient WAT organoid model, which enables accurate analysis of cellular and molecular markers of adipogenic differentiation and adipocyte physiology.

Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1-/CD34+/CD24+ Adipose Stem/Progenitor Cells.

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34-, DLK1+/CD34dim and DLK1-/CD34+ cells. We demonstrate that DLK1-/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPß are highly expressed. Further sorting of ASCs into DLK1-/CD34+/CD24- and DLK1-/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1-/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1-/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1-/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPß but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1-/CD34+/CD24+ relative to DLK1-/CD34+/CD24- subpopulations.

CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.

The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 (SPRY1) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: Insertion/Deletion; PAM: Protospacer adjacent motif; sWAT: Subcutaneous white adipose tissue; TIDE: Tracking of indels by decomposition.

Sprouty1 Prevents Cellular Senescence Maintaining Proliferation and Differentiation Capacity of Human Adipose Stem/Progenitor Cells.

The role of Ras-Mitogen-activated protein kinase (MAPK) signaling in cellular aging is not precisely understood. Recently, we identified Sprouty1 (SPRY1) as a weight-loss target gene in human adipose stem/progenitor cells (ASCs) and showed that Sprouty1 is important for proper regulation of adipogenesis. In the present study, we show that loss-of-function of Sprouty1 by CRISPR/Cas9-mediated genome editing in human ASCs leads to hyper-activation of MAPK signaling and a senescence phenotype. Sprouty1 knockout ASCs undergo an irreversible cell cycle arrest, become enlarged and stain positive for senescence-associated ß-galactosidase. Sprouty1 down-regulation leads to DNA double strand breaks, a considerably increased number of senescence-associated heterochromatin foci and induction of p53 and p21Cip1. In addition, we detect an increase of hypo-phosphorylated Retinoblastoma (Rb) protein in SPRY1 knockout ASCs. p16Ink4A is not induced. Moreover, we show that Sprouty1 knockout leads to induction of a senescence-associated secretory phenotype as indicated by the activation of the transcription factors NFκB and C/EBPß and a significant increase in mRNA expression and secretion of interleukin-8 (IL-8) and CXCL1/GROα. Finally, we demonstrate that adipogenesis is abrogated in senescent SPRY1 knockout ASCs. In conclusion, this study reveals a novel mechanism showing the importance of Sprouty1 for the prevention of senescence and the maintenance of the proliferation and differentiation capacity of human ASCs.

Fibroblast feeder layer supports adipogenic differentiation of human adipose stromal/progenitor cells.

Adipose stromal/progenitor cells (ASCs) can differentiate into adipocytes in the course of adipogenesis. This process is governed by systemic factors and signals of the adipose stem cell niche. ASCs isolated from fat tissues and amplified in vitro provide an essential and reliable model system to study adipogenesis. However, current cell culture models routinely grow ASCs on plastic surfaces largely missing niche parameters. In the present communication, we employed human foreskin fibroblasts (HFFs) monolayers as feeder cells for ASCs, which were isolated from human subcutaneous white adipose tissue and amplified in vitro. We found that PPARγ2 and several adipocyte markers were significantly higher expressed in differentiated ASCs growing on feeder layers relative to plastic dishes. Moreover, a significant higher number of adipocytes was generated from ASCs cultured on feeder layer and these adipocytes contained larger fat droplets. Insulin strongly stimulated glucose uptake into adipocytes produced on feeder layer suggesting that these cells show characteristic metabolic features of fat cells.  Finally, we show that the HFF feeder layer allows adipogenic differentiation of low-density-seeded ASCs. In conclusion, we demonstrate that the HFF feeder layer increases adipocyte differentiation of ASCs and allows differentiation of low density seeded progenitor cells  into functional adipocytes.

Reliable reference genes for expression analysis of proliferating and adipogenically differentiating human adipose stromal cells.

BACKGROUND: The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1+/CD34+/CD90+/CD105+/CD45-/CD31-. METHODS: We evaluated the applicability of 10 candidate reference genes (GAPDH, TBP, RPS18, EF1A, TFRC, GUSB, PSMD5, CCNA2, LMNA and MRPL19) using NormFinder, geNorm and BestKeeper software. RESULTS: The results indicate that EF1A and MRPL19 are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. PSMD5 serves as the most reliable endogenous control in adipogenesis. CCNA2 and LMNA were among the least consistent genes. CONCLUSIONS: Applying these findings for future gene expression analyses will help elucidate ASC biology.

Autophagy-related cell death by pan-histone deacetylase inhibition in liver cancer.

Autophagy is a homeostatic, catabolic degradation process and cell fate essential regulatory mechanism. Protracted autophagy triggers cell death; its aberrant function is responsible for several malignancies. Panobinostat, a potent pan-deacetylase inhibitor, causes endoplasmic reticulum stress-induced cell death. The aim of this study was to investigate the role of autophagy in deacetylase inhibitor-triggered liver cancer cell death.HepG2 (p53wt) and Hep3B (p53 null) liver cancer cell lines were exposed to panobinostat. RT-qPCR and western blot confirmed autophagic factor modulation. Immuno-fluorescence, -precipitation and -histochemistry as well as transmission electron microscopy verified autophagosome formation. The cytotoxicity of panobinostat and autophagy modulators was detected using a real time cell viability assay.Panobinostat induced autophagy-related factor expression and aggregation. Map1LC3B and Beclin1 were significantly over-expressed in HepG2 xenografts in nude mice treated with panobinostat for 4 weeks. Subcellular distribution of Beclin1 increased with the appearance of autophagosomes-like aggregates. Cytosolic loss of p53, in HepG2, and p73, in Hep3B cells, and a corresponding gain of their nuclear level, together with modulation of DRAM1, were observed. Autophagosome aggregation was visible after 6 h of treatment. Treatment of cells stably expressing GFP-RFPtag Map1LC3B resulted in aggregation and a fluorescence switch, thus confirming autophagosome formation and maturation. Tamoxifen, an inducer of autophagy, caused only a block in cell proliferation; but in combination with panobinostat it resulted in cell death.Autophagy triggers cell demise in liver cancer. Its modulation by the combination of tamoxifen and panobinostat could be a new option for palliative treatment of hepatocellular carcinoma.

Endoplasmic reticulum stress plays a pivotal role in cell death mediated by the pan-deacetylase inhibitor panobinostat in human hepatocellular cancer cells.

Panobinostat, a pan-deacetylase inhibitor, represents a novel therapeutic option for cancer diseases. Besides its ability to block histone deacetylases (HDACs) by promoting histone hyperacetylation, panobinostat interferes with several cell death pathways providing a potential efficacy against tumors. We have previously demonstrated that panobinostat has a potent apoptotic activity in vitro and causes a significant growth delay of hepatocellular carcinoma (HCC) tumor xenografts in nude mice models. Here, we show that treatment with panobinostat is able to induce noncanonical apoptotic cell death in HepG2 and in Hep3B cells, involving the endoplasmic reticulum (ER) stress by up-regulation of the molecular chaperone binding immunoglobulin protein/glucose-regulated protein 78, activation of eukaryotic initiation factor 2α-activating transcription factor 4 (tax-responsive enhancer element B67) and inositol requiring 1α-X-box binding protein 1 factors, strong increase and nuclear translocation of the transcription factor C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153, and involvement of c-Jun N-terminal kinase. These signaling cascades culminate into the activation of the ER-located caspase-4/12 and of executioner caspases, which finally lead to cell demise. Our results clearly show that panobinostat induces an alternative ER stress-mediated cell death pathway in HCC cells, independent of the p53 status.

Late-onset manifestation of antenatal Bartter syndrome as a result of residual function of the mutated renal Na+-K+-2Cl- co-transporter.

Genetic defects of the Na+-K+-2Cl- (NKCC2) sodium potassium chloride co-transporter result in severe, prenatal-onset renal salt wasting accompanied by polyhydramnios, prematurity, and life-threatening hypovolemia of the neonate (antenatal Bartter syndrome or hyperprostaglandin E syndrome). Herein are described two brothers who presented with hyperuricemia, mild metabolic alkalosis, low serum potassium levels, and bilateral medullary nephrocalcinosis at the ages of 13 and 15 yr. Impaired function of sodium chloride reabsorption along the thick ascending limb of Henle's loop was deduced from a reduced increase in diuresis and urinary chloride excretion upon application of furosemide. Molecular genetic analysis revealed that the brothers were compound heterozygotes for mutations in the SLC12A1 gene coding for the NKCC2 co-transporter. Functional analysis of the mutated rat NKCC2 protein by tracer-flux assays after heterologous expression in Xenopus oocytes revealed significant residual transport activity of the NKCC2 p.F177Y mutant construct in contrast to no activity of the NKCC2-D918fs frameshift mutant construct. However, coexpression of the two mutants was not significantly different from that of NKCC2-F177Y alone or wild type. Membrane expression of NKCC2-F177Y as determined by luminometric surface quantification was not significantly different from wild-type protein, pointing to an intrinsic partial transport defect caused by the p.F177Y mutation. The partial function of NKCC2-F177Y, which is not negatively affected by NKCC2-D918fs, therefore explains a mild and late-onset phenotype and for the first time establishes a mild phenotype-associated SLC12A1 gene mutation.

Influence of gain of function epithelial chloride channel ClC-Kb mutation on hearing thresholds.

Hearing depends on functional ClC-K-type chloride channels composed of barttin with ClC-Ka or ClC-Kb. Loss-of-function mutations of the barttin gene BSND or of both, the ClC-Ka gene CLNKA and the ClC-Kb gene CLNKB lead to congenital deafness and renal salt wasting. Recently, we identified the gain-of-function mutation ClC-Kb(T481S) which is associated with increased blood pressure. To explore the impact of ClC-Kb(T481S) on hearing, healthy volunteers (n=329) and individuals suffering from tinnitus (n=246) volunteered for hearing tests (n=348) and genetic analysis (n=575). 19.1% of the individuals were heterozygote (ClC-Kb(T481S)/ClC-Kb) and 1.7% homozygote carriers. Pure tone average hearing threshold (PTAt) for air conduction was significantly (p<0.033) lower in ClC-Kb(T481S) carriers (13.2+/-1.2dB) than in wild-type individuals (17.1+/-0.9dB). The prevalence of ClC-Kb(T481S) carriers was significantly increased (29.7%) in individuals with PTAt<15dB (p<0.05) and significantly decreased (13.2%) in individuals with PTAt>30 dB (p<0.017). The difference was largely due to the female population. Bone conduction was less affected pointing to an effect of the mutation on middle ear function. Tinnitus tended to be more frequent in ClC-Kb(T481S) carriers, a difference, however, not statistically significant. In conclusion, hearing thresholds are slightly lower in carriers of ClC-Kb(T481S), i.e., the gain-of-function polymorphism ClC-Kb(T481S) exerts a subtle but significant protective effect against hearing loss.

pH dependence of extracellular calcium sensing receptor activity determined by a novel technique.

BACKGROUND: Increasing evidence points to the role of the extracellular Calcium Sensing Receptor (CaSR) as a multimodal receptor responding to diverse physiologic stimuli, such as extracellular divalent and polyvalent cations, amino acids, and ionic strength. Within the kidney, these stimuli converge on the CaSR to coordinate systemic calcium and water homeostasis. In this process, the impact of urinary pH changes on the activity of the CaSR has not yet been defined. We therefore performed the present study to analyze the pH sensitivity of the CaSR. METHODS: To assess the activation state of the CaSR, we developed a new method based on the functional coupling between CaSR activity and gating of calcium sensitive potassium currents mediated by SK4 potassium channels. Two-electrode voltage clamping was used to determine whole cell currents in Xenopus oocytes heterologously expressing rat CaSR and rat SK4 potassium channels. RESULTS: Coexpression of CaSR and SK4 gave rise to potassium currents that were dependent on CaSR-mediated intracellular calcium release, and thereby corresponded to the activation state of the CaSR. In presence of extracellular calcium, ambient alkalinization above pH 7.5 increased CaSR activity. Evaluation of the CaSR calcium sensitivity at various ambient proton concentrations revealed that this effect was due to a sensitization of the CaSR towards extracellular calcium. CONCLUSION: Coexpression with SK4 potassium channels provides a fast and sensitive approach to evaluate CaSR activity in Xenopus oocytes. As disclosed by this novel technique, CaSR activity is regulated by extracellular pH.

Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases.

BACKGROUND: ClC-Ka and ClC-Kb, chloride channels participating in renal tubular Cl- transport, require the coexpression of barttin to become functional. Mutations of the barttin gene lead to the Bartter's syndrome variant BSND, characterized by congenital deafness and severe renal salt wasting. Barttin bears a proline-tyrosine motif, a target structure for the ubiquitin ligase Nedd4-2, which mediates the clearance of channel proteins from the cell membrane. Nedd4-2 is, in turn, a target of the serum- and glucocorticoid-inducible kinase SGK1, which phosphorylates and, thus, inactivates the ubiquitin ligase. ClC-Ka also possesses a SGK1 consensus site in its sequence. We hypothesized that ClC-Ka/barttin is stimulated by SGK1, and down-regulated by Nedd4-2, an effect that may be reversed by SGK1 and its isoforms, SGK2 or SGK3. METHODS: To test this hypothesis, ClC-Ka/barttin was heterologously expressed in Xenopus oocytes with or without the additional expression of Nedd4-2, SGK1, SGK2, SGK3, constitutively active S422DSGK1, or inactive K127NSGK1. RESULTS: Expression of ClC-Ka/barttin induced a slightly inwardly rectifying current that was significantly decreased upon coexpression of Nedd4-2, but not the catalytically inactive mutant C938SNedd4-2. The coexpression of S422DSGK1, SGK1, or SGK3, but not SGK2 or K127NSGK1 significantly stimulated the current. Moreover, S422DSGK1, SGK1, and SGK3 also phosphorylated Nedd4-2 and thereby inhibited Nedd4-2 binding to its target. The down-regulation of ClC-Ka/barttin by Nedd4-2 was abolished by elimination of the PY motif in barttin. CONCLUSION: ClC-Ka/barttin channels are regulated by SGK1 and SGK3, which may thus participate in the regulation of transport in kidney and inner ear.

Activating mutation of the renal epithelial chloride channel ClC-Kb predisposing to hypertension.

The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-Kb(T481S) polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-Kb(T481S) in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-Kb(T481S) were associated with significantly higher systolic (by approximately 6.0 mm Hg) and diastolic (by approximately 4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive (> or =140/90 mm Hg) blood pressure levels. Individuals carrying ClC-Kb(T481S) had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-Kb(T481S) of the renal epithelial Cl- channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.

A common sequence variation of the CLCNKB gene strongly activates ClC-Kb chloride channel activity.

BACKGROUND: Tubular transepithelial reabsorption of chloride along the nephron is a major determinant of body salt and water homeostasis and blood pressure regulation. About 40% of the glomerulary filtered sodium chloride are reabsorbed in the distal nephrons. Vectorial transepithelial sodium chloride transport is critically dependent on the function of basolateral ClC-K type chloride channels there. Modulation of ClC-Kb chloride channel activity by polymorphic variations of the CLCNKB gene, thus, could form a molecular basis for salt sensitivity of blood pressure regulation. In this study we tested the effect of several polymorphic variants on ClC-Kb chloride channel activity. METHODS: After heterologous expression in Xenopus oocytes, ClC-Kb channel activity and surface expression in presence of the ClC-K beta subunit barttin were determined by two-electrode voltage-clamp analysis, immunofluorescence, and ClC-Kb surface enzyme-linked immunosorbent assay (ELISA). RESULTS: Chloride currents induced by the ClC-Kb variants L27R, G214A, I419V, T562M, and E578K were not significantly different from wild-type currents. The ClC-KbT481S variation, however, which showed a frequency of 20% in our control population, dramatically activated chloride conductance by a factor of 20. Activation of chloride currents was also observed after introducing homologous mutations in ClC-Ka and ClC-K1, but not in ClC-2 and ClC-5 chloride channels. ClC-Kb activation by the T481S mutation did not change intrinsic ion channel pore properties and did not require increased surface expression of ClC-KbT481S. CONCLUSION: Genetic heterogeneity of ClC-Kb chloride channels correlates with functional heterogeneity, which assigns ClC-Kb to a set of genes potentially relevant for polygenic salt-sensitivity of blood pressure regulation.