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Aim: Impaired calcium homeostasis limits life expectancy and quality in familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC). This rare disease is caused by loss-of-function mutations in CLDN16 or CLDN19 genes leading to impaired paracellular reabsorption of divalent cations along the cortical thick ascending limb (cTAL). The ensuing late distal nephron and collecting duct system partially compensate for the defect in cTAL by increased transcellular Ca2+ reabsorption via the luminal transient receptor potential channel V5 (TRPV5), as well as basolateral plasma membrane calcium ATPase (PMCA) and sodium-potassium exchanger (NCX1). The loop diuretic furosemide induces compensatory activation in these distal segments due to blockade of NaCl and divalent cation reabsorption in the preceeding TAL. Although furosemide enhances urinary calcium excretion via inhibition of the aforementioned cTAL in general population, this effect is not expected in FHHNC patients with already severely impaired Ca2+ transport in the cTAL. The present study follows the hypothesis that furosemide may alleviate hypercalciuria in this disease by activation of the distal transcellular Ca2+ transport proteins. Methods: Cldn16-deficient mice (Cldn16-/- ) served as a FHHNC model. Wild-type (WT) and Cldn16-/- mice were treated with furosemide (7 days of 40 mg/kg bw) or vehicle. We assessed renal electrolyte handling (metabolic cages) and key divalent transport proteins. Results: Cldn16-/- mice show increased Ca2+ excretion than WT and compensatory stimulation of Cldn2 in the proximal tubule, as well as of TRPV5, and NCX1 in the distal nephron at baseline. Furosemide reduced hypercalciuria in Cldn16-/- mice and enhanced TRPV5 and PMCA levels in Cldn16-/- but not in WT mice. Conclusions: Furosemide alleviated hypercalciuria in a mouse FHHNC model, likely via stimulation of transcellular luminal and basolateral Ca2+ transport systems in the distal nephron and collecting duct. These results may have a clinical implication in FHHNC patients. No conflict of interests This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Claudins are a family of transmembrane proteins expressed in epithelial tissues and are the major components of tight junctions (TJs), which define barrier properties in epithelia and maintain cell polarity. How claudins regulate the formation of TJs and which functions they exert outside of them is not entirely understood. Although the long and unstructured C-terminal tail is essential for regulation, it is unclear how it is involved in these functions beyond interacting with TJ-associated proteins such as TJ protein ZO-1 (TJP1). Here, we present an interactome study of the pan-claudin family in Madin-Darby canine kidney (MDCK)-C7 cells by combining two complementary mass spectrometry-based pull-down techniques creating an interaction landscape of the entire claudin family. The interaction partners of the claudins' C termini reveal their possible implications in localized biological processes in epithelial cells and their regulation by post-translational modifications (PTMs).

Claudin-10b is an important component of the tight junction in the thick ascending limb (TAL) of Henle's loop and allows paracellular sodium transport. In immunofluorescence stainings, claudin-10b–positive cells exhibited extensive extra staining of basolateral, column-like structures. The precise localization and function have so far remained elusive. In isolated cortical TAL segments from C57BL/6J mice, kidney-specific claudin-10 knockout mice (cKO), and respective litter mates (WT), we investigated the localization and protein expression and function by fluorescence microscopy and electrophysiological measurements. Ultrastructural analysis of TAL in kidney sections was performed by electron microscopy. Claudin-10b colocalized with the basolateral Na+-K+ ATPase and the Cl– channel subunit barttin, but the lack of claudin-10b did not influence the localization or abundance of these proteins. However, the accessibility of the basolateral infolded extracellular space to ouabain or fluorescein was increased by basolateral Ca2+ removal and in the absence of claudin-10b. Ultrastructural analysis by electron microscopy revealed a widening of basolateral membrane infoldings in cKO in comparison to WT. We hypothesize that claudin-10b shapes neighboring membrane invaginations by trans interaction to stabilize and facilitate high-flux salt transport in a water-tight epithelium.

BACKGROUND: The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiological role of claudin-10a in the kidney has been unclear. METHODS: To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice; confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining; and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. RESULTS: Mice deficient in claudin-10a were fertile and without overt phenotypes. Upon knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a consequence, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison of other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, as well as unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. CONCLLUSIONS: Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyperreabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.

Annals of the New York Academy of Sciences 1516(1), 197-211 (2022). doi:10.1111/nyas.14865, In epithelia, claudin proteins are important components of the tight junctions as they determine the permeability and specificity to ions of the paracellular pathway. Mutations in CLDN10 cause the rare autosomal recessive HELIX syndrome (Hypohidrosis, Electrolyte imbalance, Lacrimal gland dysfunction, Ichthyosis, and Xerostomia), in which patients display severe enamel wear. Here, we assess whether this enamel wear is caused by an innate fragility directly related to claudin-10 deficiency in addition to xerostomia. A third molar collected from a female HELIX patient was analyzed by a combination of microanatomical and physicochemical approaches (i.e., electron microscopy, elemental mapping, Raman microspectroscopy, and synchrotron-based X-ray fluorescence). The enamel morphology, formation time, organization, and microstructure appeared to be within the natural variability. However, we identified accentuated strontium variations within the HELIX enamel, with alternating enrichments and depletions following the direction of the periodical striae of Retzius. These markings were also present in dentin. These data suggest that the enamel wear associated with HELIX may not be related to a disruption of enamel microstructure but rather to xerostomia. However, the occurrence of events of strontium variations within dental tissues might indicate repeated episodes of worsening of the renal dysfunction that may require further investigations., Published by New York Acad. of Sciences, New York, NY

International audience; Functional properties of the paracellular pathway depend critically on the set of claudins (CLDN) expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX) syndrome caused by genetic variations in the CLDN10 gene and familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by genetic variations in the CLDN16 or CLDN19 genes. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, CLDN16, and CLDN19 in both paraffin-embedded and frozen kidney sections from the adult human, mouse, and rat using immunohistochemistry and immunofluorescence, respectively. Here, CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junctions in human and rodent kidneys. Weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in the cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidneys. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of the TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junctions almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidneys.NEW & NOTEWORTHY This study used immunohistochemistry and immunofluorescence to investigate the distribution of claudin 10, 16, and 19 in the human, mouse, and rat kidney. The findings showed distinct junctional pathways in both human and rodent kidneys, supporting the existence of different permeation profiles in all species investigated.

Patients with mutations in Cyclin M2 (CNNM2) suffer from hypomagnesaemia, seizures, and intellectual disability. Although the molecular function of CNNM2 is under debate, the protein is considered essential for renal Mg2+ reabsorption. Here, we used a Cnnm2 knock out mouse model, generated by CRISPR/Cas9 technology, to assess the role of CNNM2 in Mg2+ homeostasis. Breeding Cnnm2+/− mice resulted in a Mendelian distribution at embryonic day 18. Nevertheless, only four Cnnm2−/− pups were born alive. The Cnnm2−/− pups had a significantly lower serum Mg2+ concentration compared to wildtype littermates. Subsequently, adult Cnnm2+/− mice were fed with low, control, or high Mg2+ diets for two weeks. Adult Cnnm2+/− mice showed mild hypomagnesaemia compared to Cnnm2+/+ mice and increased serum Ca2+ levels, independent of dietary Mg2+ intake. Faecal analysis displayed increased Mg2+ and Ca2+ excretion in the Cnnm2+/− mice. Transcriptional profiling of Trpm6, Trpm7, and Slc41a1 in kidneys and colon did not reveal effects based on genotype. Microcomputed tomography analysis of the femurs demonstrated equal bone morphology and density. In conclusion, CNNM2 is vital for embryonic development and Mg2+ homeostasis. Our data suggest a previously undescribed role of CNNM2 in the intestine, which may contribute to the Mg2+ deficiency in mice and patients.

The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle's loop in the kidney. In patients, mutations in CLDN16 cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10 impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin.

Phosphatases of regenerating liver (PRLs), the most oncogenic of all protein-tyrosine phosphatases (PTPs), play a critical role in metastatic progression of cancers. Recent findings established a new paradigm by uncovering that their association with magnesium transporters of the cyclin M (CNNM) family causes a rise in intracellular magnesium levels that promote oncogenic transformation. Recently, however, essential roles for regulation of the circadian rhythm and reproduction of the CNNM family have been highlighted. Here, we describe the crystal structure of PRL-1 in complex with the Bateman module of CNNM2 (CNNM2BAT), which consists of two cystathionine β-synthase (CBS) domains (IPR000664) and represents an intracellular regulatory module of the transporter. The structure reveals a heterotetrameric association, consisting of a disc-like homodimer of CNNM2BAT bound to two independent PRL-1 molecules, each one located at opposite tips of the disc. The structure highlights the key role played by Asp-558 at the extended loop of the CBS2 motif of CNNM2 in maintaining the association between the two proteins and proves that the interaction between CNNM2 and PRL-1 occurs via the catalytic domain of the phosphatase. Our data shed new light on the structural basis underlying the interaction between PRL phosphatases and CNNM transporters and provides a hypothesis about the molecular mechanism by which PRL-1, upon binding to CNNM2, might increase the intracellular concentration of Mg2+ thereby contributing to tumor progression and metastasis. The availability of this structure sets the basis for the rational design of compounds modulating PRL-1 and CNNM2 activities.

Background In Inflammatory bowel disease, intestinal homeostasis cannot be sustained due to an overreaction of the immune system and a disturbed epithelial barrier due to an impaired tight junction (TJ). At tricellular TJs (tTJs), which are formed where three cells meet, the TJ network is basolaterally expanded and forms a central tube which would be potentially large enough to allow for a paracellular passage of macromolecules. Under normal conditions, the tTJ protein tricellulin (Tric) seals the central tube against luminal uptake. However, in ulcerative colitis (UC), Tric is downregulated, resulting in increased uptake of macromolecules including antigens which may further support the inflammatory processes. Thus, we aimed to identify whether or not overexpression of Tric has a protective effect against inflammation. Methods We generated intestinal epithelial cell-specific overexpression of Tric in mice (B6.C-Tg(Vil-cre) Rosa26tgGFP-Tric). In Tric-overexpressing (Tric-OE) and in wild-type (wt) mice experimental colitis was induced by 3% dextran sodium sulphate (DSS) for 6 days. The effect was assessed by determining the disease activity index (DAI: weight loss, stool consistency, and stool bleeding) and the colon dimensions. Using chamber experiments were performed to determine permeabilities for ions and macromolecules of different sizes (4 and 10 kDa). Results No difference in weight loss was observed between wt and Tric-OE mice upon DSS treatment. However, Tric-OE mice suffered less from diarrhoea and had less bloody stools compared with wt mice resulting in a lower DAI. In addition, DSS caused a reduced colon length which was less prominent in Tric-OE mice than in wt mice. Functional analysis revealed higher permeabilities for Na+ and Cl− after DSS treatment with no significant differences between Tric-OE and wt mice. In contrast, permeabilities for macromolecules of both sizes were unchanged after DSS treatment in Tric-OE mice but elevated in wt-mice. Conclusion Tric-overexpression prevents the epithelial barrier from elevated paracellular uptake of macromolecules during DSS colitis without affecting ion permeability. This protects against DSS colitis as indicated by less affected clinical and structural outcome parameters, most likely via a reduction in a load of immunologically active macromolecules in the subepithelium.

Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients.

SORCS1 and SORCS3 are two related sorting receptors expressed in neurons of the arcuate nucleus of the hypothalamus. Using mouse models with individual or dual receptor deficiencies, we document a previously unknown function of these receptors in central control of metabolism. Specifically, SORCS1 and SORCS3 act as intracellular trafficking receptors for tropomyosin‐related kinase B to attenuate signaling by brain‐derived neurotrophic factor, a potent regulator of energy homeostasis. Loss of the joint action of SORCS1 and SORCS3 in mutant mice results in excessive production of the orexigenic neuropeptide agouti‐related peptide and in a state of chronic energy excess characterized by enhanced food intake, decreased locomotor activity, diminished usage of lipids as metabolic fuel, and increased adiposity, albeit at overall reduced body weight. Our findings highlight a novel concept in regulation of the melanocortin system and the role played by trafficking receptors SORCS1 and SORCS3 in this process.

The thick ascending limb (TAL) of Henle's loop drives paracellular Na+, Ca2+, and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins that consist of four transmembrane segments, two extracellular segments (ECS1 and -2), and one intracellular loop. Claudins interact within the same (cis) and opposing (trans) plasma membranes. The claudins Cldn10b, -16, and -19 facilitate cation reabsorption in the TAL, and their absence leads to a severe disturbance of renal ion homeostasis. We combined electrophysiological measurements on microperfused mouse TAL segments with subsequent analysis of claudin expression by immunostaining and confocal microscopy. Claudin interaction properties were examined using heterologous expression in the TJ-free cell line HEK 293, live-cell imaging, and Forster/FRET. To reveal determinants of interaction properties, a set of TAL claudin protein chimeras was created and analyzed. Our main findings are that (i) TAL TJs show a mosaic expression pattern of either cldn10b or cldn3/cldn16/cldn19 in a complex; (ii) TJs dominated by cldn10b prefer Na+ over Mg2+, whereas TJs dominated by cldn16 favor Mg2+ over Na+; (iii) cldn10b does not interact with other TAL claudins, whereas cldn3 and cldn16 can interact with cldn19 to form joint strands; and (iv) further claudin segments in addition to ECS2 are crucial for trans interaction. We suggest the existence of at least two spatially distinct types of paracellular channels in TAL: a cldn10b-based channel for monovalent cations such as Na+ and a spatially distinct site for reabsorption of divalent cations such as Ca2+ and Mg2.

Introduction La branche large ascendante corticale de l’anse de Henle (BLAHC) reabsorbe 25 % du calcium filtre, exclusivement par voie paracellulaire ; le voltage transepithelial (Vte) lumiere-positive constitue la force motrice de la reabsorption. Vte depend de la secretion paracellulaire du sodium. L’expression a la jonction serree de la claudine (Cldn) 10b rend la permeabilite paracellulaire au sodium (PNa) superieure a celle du chlore (PCl). L’hormone parathyroidienne (PTH) augmente la reabsorption de calcium dans la BLAHC mais les mecanismes sont mal connus. Methodes L’hypothese testee est que l’effet de la PTH dans la BLAHC implique la Cldn10b qui determine Vte et PNa/PCl. Des BLAHC de souris Cldn10b+/+ et Cldn10b−/− ont ete microperfusees in vitro pour mesurer Vte et PNa/PCl a l’etat basal, et/ou apres ajout de PTH (10−10 M), de phorbol 12-Myristate 13-Acetate (PMA 10−6 M), de dibutyryl cAMP (dbcAMP 5 10−4 M), de ionomycine (10−7 M), de PKI 14–22 amide (inhibiteur de proteine kinase A PKA 10−6 M), de trifluoroperazine (TFP inhibiteur de la calcium-calmoduline kinase 10−4 M), de Dyngo4a (inhibiteur de l’endocytose mediee par la clathrine 10−5 M). Le rapport PNa/PCl est calcule par l’equation de Goldman-Hodgkin-Katz. Resultats obtenus ou attendus La PTH augmente PNa/PCl dans des BLAHC de souris Cldn10b+/+ (p = 0,0002) mais pas dans celles de souris Cldn10−/−. Cet effet est reproduit par le dbcAMP (p = 0,02) et la ionomycine (p = 0,047), mais pas par le PMA. Le PKI, la TFP et le Dyngo4a inhibent l’effet de la PTH sur PNa/PCl. Conclusion La PTH augmente PNa/PCl via la Cldn10b en activant la PKA et la calcium-calmoduline kinase. L’inhibition de l’endocytose mediee par la clathrine previent cet effet. Les proprietes des jonctions serrees intercellulaires peuvent etre directement controlees par des voies de signalisation intracellulaire affectant le trafic intracellulaire.

Claudins are tight junction membrane proteins and regulate the paracellular passage of ions and water. They can seal the paracellular cleft against solute passage but also form paracellular channels. They are tetraspan proteins with two extracellular segments. Claudin-10 exists in at least two functional isoforms, claudin-10a and claudin-10b, that differ in their first transmembrane segment and first extracellular segment. Both isoforms act as selective paracellular ion channels, either for anions (claudin-10a) or for cations (claudin-10b). Their diverse functions are reflected in completely different expression patterns in the body, especially in the kidney. Their structural and functional similarities and differences make them ideal subjects to study determinants of claudin charge selectivity and pore formation. This review aims to summarise research on permeability properties of the claudin-10 channels and their role in physiology and pathophysiology of the kidney.

In the kidney, tight junction proteins contribute to segment specific selectivity and permeability of paracellular ion transport. In the thick ascending limb (TAL) of Henle's loop, chloride is reabsorbed transcellularly, whereas sodium reabsorption takes transcellular and paracellular routes. TAL salt transport maintains the concentrating ability of the kidney and generates a transepithelial voltage that drives the reabsorption of calcium and magnesium. Thus, functionality of TAL ion transport depends strongly on the properties of the paracellular pathway. To elucidate the role of the tight junction protein claudin-10 in TAL function, we generated mice with a deletion of Cldn10 in this segment. We show that claudin-10 determines paracellular sodium permeability, and that its loss leads to hypermagnesemia and nephrocalcinosis. In isolated perfused TAL tubules of claudin-10–deficient mice, paracellular permeability of sodium is decreased, and the relative permeability of calcium and magnesium is increased. Moreover, furosemide-inhibitable transepithelial voltage is increased, leading to a shift from paracellular sodium transport to paracellular hyperabsorption of calcium and magnesium. These data identify claudin-10 as a key factor in control of cation selectivity and transport in the TAL, and deficiency in this pathway as a cause of nephrocalcinosis.

Neurotrophins are essential for development and maintenance of the vertebrate nervous system. Paradoxically, although mature neurotrophins promote neuronal survival by binding to tropomyosin receptor kinases and p75 neurotrophin receptor (p75(NTR)), pro-neurotrophins induce apoptosis in cultured neurons by engaging sortilin and p75(NTR) in a death-signaling receptor complex. Substantial amounts of neurotrophins are secreted in pro-form in vivo, yet their physiological significance remains unclear. We generated a sortilin-deficient mouse to examine the contribution of the p75(NTR)/sortilin receptor complex to neuronal viability. In the developing retina, Sortilin 1 (Sort1)(-/-) mice showed reduced neuronal apoptosis that was indistinguishable from that observed in p75(NTR)-deficient (Ngfr(-/-)) mice. To our surprise, although sortilin deficiency did not affect developmentally regulated apoptosis of sympathetic neurons, it did prevent their age-dependent degeneration. Furthermore, in an injury protocol, lesioned corticospinal neurons in Sort1(-/-) mice were protected from death. Thus, the sortilin pathway has distinct roles in pro-neurotrophin-induced apoptotic signaling in pathological conditions, but also in specific stages of neuronal development and aging. Udgivelsesdato: 2007-Nov

The proneurotrophin receptor sortilin is a protein with dual functions, being involved in intracellular protein transport, as well as cellular signal transduction. The relevance of the receptor for various neuronal disorders, such as dementia, seizures, and brain injury, is well established. In contrast, little is known about the role of sortilin in immune cells and inflammatory diseases. The aim of our study was to elucidate the distribution of sortilin in different immune cell types in mice and humans and to analyze its function in autoimmune CNS inflammation. Sortilin was expressed most profoundly in murine and human macrophages and dendritic cells and to a much lesser extent in B and T cells. In dendritic cells, sortilin had an impact on Ag processing. Accordingly, sortilin was highly expressed by infiltrated perivascular myeloid cells, mainly in vessel cuffs, in the CNS of patients suffering from multiple sclerosis, the most common inflammatory autoimmune disease of the CNS. Yet, sortilin gene-targeted mice (Sort1−/−) and chimeras deficient in sortilin in the immune system were as susceptible as wild-type littermates to T cell–dependent experimental autoimmune encephalomyelitis. Considering our results and recent data from other investigators, we conclude that the proneurotrophin receptor sortilin plays a role in innate, rather than in adaptive, immune processes and, thus, not in autoimmune neuroinflammation.

Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg / animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter. Corticosteroid binding globulin (CBG) is the major transport protein for glucocorticoids in plasma of mammalian species, with more than 90% of circulating corticosteroid mole

sorLA (sorting protein-related receptor) is a type-1 membrane protein of unknown function that is expressed in neurons. Its homology to sorting receptors that shuttle between the plasma membrane, endosomes, and the Golgi suggests a related function in neuronal trafficking processes. Because expression of sorLA is reduced in the brain of patients with Alzheimer's disease (AD), we tested involvement of this receptor in intracellular transport and processing of the amyloid precursor protein (APP) to the amyloid β-peptide (Aβ), the principal component of senile plaques. We demonstrate that sorLA interacts with APP in vitro and in living cells and that both proteins colocalize in endosomal and Golgi compartments. Overexpression of sorLA in neurons causes redistribution of APP to the Golgi and decreased processing to Aβ, whereas ablation of sorLA expression in knockout mice results in increased levels of Aβ in the brain similar to the situation in AD patients. Thus, sorLA acts as a sorting receptor that protects APP from processing into Aβ and thereby reduces the burden of amyloidogenic peptide formation. Consequently, reduced receptor expression in the human brain may increase Aβ production and plaque formation and promote spontaneous AD.

SORCS3 is an orphan receptor of the VPS10P domain receptor family, a group of sorting and signaling receptors central to many pathways in control of neuronal viability and function. SORCS3 is highly expressed in the CA1 region of the hippocampus, but the relevance of this receptor for hippocampal activity remained absolutely unclear. Here, we show that SORCS3 localizes to the postsynaptic density and that loss of receptor activity in gene-targeted mice abrogates NMDA receptor-dependent and -independent forms of long-term depression (LTD). Consistent with a loss of synaptic retraction, SORCS3-deficient mice suffer from deficits in behavioral activities associated with hippocampal LTD, particularly from an accelerated extinction of fear memory. A possible molecular mechanism for SORCS3 in synaptic depression was suggested by targeted proteomics approaches that identified the ability of SORCS3 to functionally interact with PICK1, an adaptor that sorts glutamate receptors at the postsynapse. Faulty localization of PICK1 in SORCS3-deficient neurons argues for altered glutamate receptor trafficking as the cause of altered synaptic plasticity in the SORCS3-deficient mouse model. In conclusion, our studies have identified a novel function for VPS10P domain receptors in control of synaptic depression and suggest SORCS3 as a novel factor modulating aversive memory extinction.

SummaryRecent genome-wide association studies (GWAS) have revealed strong association of hypercholesterolemia and myocardial infarction with SNPs on human chromosome 1p13.3. This locus covers three genes: SORT1, CELSR2, and PSRC1. We demonstrate that sortilin, encoded by SORT1, is an intracellular sorting receptor for apolipoprotein (apo) B100. It interacts with apoB100 in the Golgi and facilitates the formation and hepatic export of apoB100-containing lipoproteins, thereby regulating plasma low-density lipoprotein (LDL) cholesterol. Absence of sortilin in gene-targeted mice reduces secretion of lipoproteins from the liver and ameliorates hypercholesterolemia and atherosclerotic lesion formation in LDL receptor-deficient animals. In contrast, sortilin overexpression stimulates hepatic release of lipoproteins and increases plasma LDL levels. Our data have uncovered a regulatory pathway in hepatic lipoprotein export and suggest a molecular explanation for the cardiovascular risk being associated with 1p13.3.

Claudin-16 (CLDN16) is critical for renal paracellular epithelial transport of Ca2+and Mg2+in the thick ascending loop of Henle. To gain novel insights into the role of CLDN16 in renal Ca2+and Mg2+homeostasis and the pathological mechanisms underlying a human disease associated with CLDN16 dysfunction [familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), OMIM 248250], we generated a mouse model of CLDN16 deficiency. Similar to patients, CLDN16-deficient mice displayed hypercalciuria and hypomagnesemia. Contrary to FHHNC patients, nephrocalcinosis was absent in our model, indicating the existence of compensatory pathways in ion handling in this model. In line with the renal loss of Ca2+, compensatory mechanisms like parathyroid hormone and 1,25(OH)2D3were significantly elevated. Also, gene expression profiling revealed transcriptional upregulation of several Ca2+and Mg2+transport systems including Trpv5, Trpm6, and calbindin-D9k. Induced gene expression was also seen for the transcripts of two putative Mg2+transport proteins, Cnnm2 and Atp13a4. Moreover, urinary pH was significantly lower when compared with wild-type mice. Taken together, our findings demonstrate that loss of CLDN16 activity leads to specific alterations in Ca2+and Mg2+homeostasis and that CLDN16-deficient mice represent a useful model to further elucidate pathways involved in renal Ca2+and Mg2+handling.

ClC-4 and ClC-5, together with ClC-3, form a distinct branch of the CLC chloride channel family. Although ClC-5 was shown to be mainly expressed in endocytotic vesicles, expression of ClC-5 in Xenopus oocytes elicited chloride currents. We now show that ClC-4 also gives rise to strongly outwardly rectifying anion currents when expressed in oocytes. They closely resemble ClC-5 currents with which they share a NO3- > Cl- > Br- > I- conductance sequence that differs from that reported for the highly homologous ClC-3. Both ClC-4 and ClC-5 currents are reduced by lowering extracellular pH. We could measure similar currents after expressing either channel in HEK293 cells. To demonstrate that these currents are directly mediated by the channel proteins, we introduced several point mutations that change channel characteristics. In ClC-5, several point mutations alter the kinetics of activation but leave macroscopic rectification and ion selectivity unchanged. A mutation (N565K) equivalent to a mutation reported to have profound effects on ClC-3 does not have similar effects on ClC-5. Moreover, a mutation at the end of D2 (S168T in ClC-5) changes ion selectivity, and a mutation at the end of D3 (E211A in ClC-5 and E224A in ClC-4) changes voltage dependence and ion selectivity. This shows that ClC-4 and ClC-5 can directly mediate plasma membrane currents.

SummaryImmunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules.