Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl− concentration. Disruption of endosomal ClC‐3 causes severe neurodegeneration. To assess the importance of ClC‐3 Cl−/H+ exchange, we now generate Clcn3unc/unc mice in which ClC‐3 is converted into a Cl− channel. Unlike Clcn3−/− mice, Clcn3unc/unc mice appear normal owing to compensation by ClC‐4 with which ClC‐3 forms heteromers. ClC‐4 protein levels are strongly reduced in Clcn3−/−, but not in Clcn3unc/unc mice because ClC‐3unc binds and stabilizes ClC‐4 like wild‐type ClC‐3. Although mice lacking ClC‐4 appear healthy, its absence in Clcn3unc/unc/Clcn4−/− mice entails even stronger neurodegeneration than observed in Clcn3−/− mice. A fraction of ClC‐3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3unc/unc or Clcn3−/− mice before neurodegeneration sets in. Both, Cl−/H+‐exchange activity and the stabilizing effect on ClC‐4, are central to the biological function of ClC‐3

Tissue distribution of Kir7.1 inwardly rectifying K⁺ channel probed in a knock-in mouse expressing a haemagglutinin-tagged protein

Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It might therefore become a useful tool to unravel Kir7.1 function in the different organs where it is expressed

Cleft Palate, Moderate Lung Developmental Retardation and Early Postnatal Lethality in Mice Deficient in the Kir7.1 Inwardly Rectifying K+ Channel.

Kir7.1 is an inwardly rectifying K+ channel of the Kir superfamily encoded by the kcnj13 gene. Kir7.1 is present in epithelial tissues where it colocalizes with the Na+/K+-pump probably serving to recycle K+ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree

Double Knockout of the Na+-Driven Cl-/HCO3--Exchanger and Na+/Cl- Cotransporter Induces Hypokalemia and Volume Depletion

We recently described a novel thiazide-sensitive electroneutral NaCI transport mechanism resulting from the parallel operation of the Cl-/HCO3- exchanger pendrin and the Na+-driven Cl-/2HCO(3)(-) exchanger (NDCBE) in beta-intercalated cells of the collecting duct. Although a role for pendrin in maintaining Na+ balance, intravascular volume, and BP is well supported, there is no in vivo evidence for the role of NDCBE in maintaining Na+ balance. Here, we show that deletion of NDCBE in mice caused only subtle perturbations of Na homeostasis and provide evidence that the Na+/Cl- cotransporter (NCC) compensated for the inactivation of NDCBE. To unmaskthe role of NDCBE, we generated Ndcbe/Ncdclouble-knockout (dKO) mice. On a normal salt diet, dKO and single-knockout mice exhibited similar activation of the renin-angiotensin-aldosterone system, whereas only dKO mice displayed a lower blood K concentration. Furthermore, dKO mice displayed upregulation of the epithelial sodium channel (ENaC) and the Ca2+-activated K+ channel BKCa. During NaCI depletion, only dKO mice developed marked intravascular volume contraction, despite dramatically increased renin activity. Notably, the increase in aldosterone levels expected on NaCI depletion was attenuated in dKO mice, and single-knockout and dKO mice had similar blood K+ concentrations under this condition. In conclusion, NDCBE is necessary for maintaining sodium balance and intravascular volume during salt depletion or NCC inactivation in mice. Furthermore, NDCBE has an important role in the prevention of hypokalemia. Because NCC and NDCBE are both thiazide targets, the combined inhibition of NCC and the NDCBE/pendrin system may explain thiazide-induced hypokalemia in some patients

Survival of <i>Kcnj13</i>^- mutant mice.

<p>Survival of <i>Kcnj13</i>^- mutant mice.</p

Basolateral expression of Kir7.1 channel in the epithelium of the airways.

<p>Immunohistochemical detection of Kir7.1 channel in trachea (left) and bronchiole (right) in adult <i>Kcnj13</i>^+/+, newborn <i>Kcnj13</i>^+/+ or newborn <i>Kcnj13</i>^-/- mice. Tissue sections were treated with anti-Kir7.1 antibody (1:15,000). Kir7.1 expression was restricted to the basolateral membrane of airway epithelium in adult and newborn <i>Kcnj13</i>^+/+ mice. Staining in <i>Kcnj13</i>^-/- tissues shows complete absence of specific immunoreactive signal. Nuclei were counterstained with Fast Red. Scale bar represents 50 μm.</p

Pulmonary abnormalities in embryonic lungs from <i>Kcnj13</i>^-/- mice.

<p>a. Hematoxylin and eosin stained lung sections taken at various gestational stages as indicated. Morphological differences in KO lungs were observed at E18.5 and P0. Null mutant mice show a lower air space and thicker walls at lung terminal sacs compared to WT and heterozygous mice. No differences were visible between <i>Kcnj13</i>^+/+ and <i>Kcnj13</i>^+/- genotypes. Scale bars represent 100 μm. b. Morphometric analysis of terminal sac spaces in lungs at various gestational stages. Significant reduction in spaces was observed in Kir7.1 deficient mice from E18.5 onwards. Results are expressed as mean ± S.E.M, # p<0.05 and * p<0.01 for the difference with WT by ANOVA. c. Graphical representation of newborn lung flotation test. Grey sections of columns correspond to percent of floating lungs, with black being the percent sinking lungs.</p

Morphology and body weights of <i>Kcnj13</i> null mutant mice.

<p>a. Analysis of Kir7.1 expression in WT, heterozygous and null mutant mice; cyclophilin A (Cyc1) is used as constitutively expressed control gene. b. Gross morphology of WT, and heterozygous and homozygous <i>Kcnj13</i> null mutant newborn pups. c. Body weight vs. embryonic stage for WT (circles), <i>Kcnj13</i>^<i>+/-</i> (triangles), and <i>Kcnj13</i>^-/- (squares) embryos. Results are expressed as mean ± S.E.M. of the following numbers of embryos: 12.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 9, <i>Kcnj13</i>^-/- 4; 13.5 dpc: WT 9, <i>Kcnj13</i>^<i>+/-</i> 14, <i>Kcnj13</i>^-/- 2; n 14.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 11, <i>Kcnj13</i>^-/- 6; 15.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 20, <i>Kcnj13</i>^-/- 14; 16.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 4; 17.5 dpc:WT 5, <i>Kcnj13</i>^<i>+/-</i> 12, <i>Kcnj13</i>^-/- 6; 18.5 dpc: WT 4, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 6; P0: WT 7, <i>Kcnj13</i>^<i>+/-</i> 15, <i>Kcnj13</i>^-/- 10. * p< 0.001; ** p <0.05 for the differences between <i>Kcnj13</i>^-/- and <i>Kcnj13</i>^<i>+/+</i> data (ANOVA).</p

SLC26A1 is a major determinant of sulfate homeostasis in humans

Sulfate plays a pivotal role in numerous physiological processes in the human body, including bone and cartilage health. A role of the anion transporter SLC26A1 (Sat1) for sulfate reabsorption in the kidney is supported by the observation of hyposulfatemia and hypersulfaturia in Slc26a1-knockout mice. The impact of SLC26A1 on sulfate homeostasis in humans remains to be defined. By combining clinical genetics, functional expression assays, and population exome analysis, we identify SLC26A1 as a sulfate transporter in humans and experimentally validate several loss-of-function alleles. Whole-exome sequencing from a patient presenting with painful perichondritis, hyposulfatemia, and renal sulfate wasting revealed a homozygous mutation in SLC26A1, which has not been previously described to the best of our knowledge. Whole-exome data analysis of more than 5,000 individuals confirmed that rare, putatively damaging SCL26A1 variants were significantly associated with lower plasma sulfate at the population level. Functional expression assays confirmed a substantial reduction in sulfate transport for the SLC26A1 mutation of our patient, which we consider to be novel, as well as for the additional variants detected in the population study. In conclusion, combined evidence from 3 complementary approaches supports SLC26A1 activity as a major determinant of sulfate homeostasis in humans. In view of recent evidence linking sulfate homeostasis with back pain and intervertebral disc disorder, our study identifies SLC26A1 as a potential target for modulation of musculoskeletal health