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ClC‐K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC‐Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline‐tyrosine motif in the C‐terminus of barttin stimulates ClC‐K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co‐expressing ClC‐K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC‐K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock‐in mouse. Comparing wild‐type (WT) and knock‐in mice under a standard diet, we could not observe any difference in ClC‐K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high‐sodium low‐potassium diet, known to induce hyperplasia of distal convoluted tubules, knock‐in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. Key points: It is revealed by mutagenesis and functional experiments that a previously identified proline‐tyrosine motif regulating ClC‐K plasma membrane levels is indeed an endocytic YxxØ motif.Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC‐K channels.Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo.Knock‐in mice with a mutation in this motif, under conditions of a high‐sodium low‐potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild‐type animals, indicating a gain of function of the channel in vivo. [ABSTRACT FROM AUTHOR]

The function of the chloride channel ClC‐1 is crucial for the control of muscle excitability. Thus, reduction of ClC‐1 functions by CLCN1 mutations leads to myotonia congenita. Many different animal models have contributed to understanding the myotonia pathophysiology. However, these models do not allow in vivo screening of potentially therapeutic drugs, as the zebrafish model does. In this work, we identified and characterized the two zebrafish orthologues (clc‐1a and clc‐1b) of the ClC‐1 channel. Both channels are mostly expressed in the skeletal muscle as revealed by RT‐PCR, western blot, and electrophysiological recordings of myotubes, and clc‐1a is predominantly expressed in adult stages. Characterization in Xenopus oocytes shows that the zebrafish channels display similar anion selectivity and voltage dependence to their human counterparts. However, they show reduced sensitivity to the inhibitor 9‐anthracenecarboxylic acid (9‐AC), and acidic pH inverts the voltage dependence of activation. Reduction of clc‐1a/b expression hampers spontaneous and mechanically stimulated movement, which could be reverted by expression of human ClC‐1 but not by some ClC‐1 containing myotonia mutations. Treatment of clc‐1‐depleted zebrafish with mexiletine, a typical drug used in human myotonia, improves the motor behaviour. Our work extends the repertoire of ClC channels to evolutionary structure–function studies and proposes the zebrafish clcn1 crispant model as a simple tool to find novel therapies for myotonia. Key points: We have identified two orthologues of ClC‐1 in zebrafish (clc‐1a and clc‐1b) which are mostly expressed in skeletal muscle at different developmental stages.Functional characterization of the activity of these channels reveals many similitudes with their mammalian counterparts, although they are less sensitive to 9‐AC and acidic pH inverts their voltage dependence of gating.Reduction of clc‐1a/b expression hampers spontaneous and mechanically stimulated movement which could be reverted by expression of human ClC‐1.Myotonia‐like symptoms caused by clc‐1a/b depletion can be reverted by mexiletine, suggesting that this model could be used to find novel therapies for myotonia. [ABSTRACT FROM AUTHOR]

Psychiatric disorders have been hypothesized to originate during development, with genetic and environmental factors interacting in the etiology of disease. Therefore, developmentally regulated genes have received attention as risk modulators in psychiatric diseases. Reelin is an extracellular protein essential for neuronal migration and maturation during development, and its expression levels are reduced in psychiatric disorders. Interestingly, several perinatal insults that increase the risk of behavioral deficits alter Reelin signaling. However, it is not known whether a dysfunction in Reelin signaling during perinatal stages increases the risk of psychiatric disorders. Here we used a floxed dab1 allele to study whether a transient decrease in Dab1, a key component of the Reelin pathway, is sufficient to induce behavioral deficits related to psychiatric disorders. We found that transient Dab1 downregulation during perinatal stages leads to permanent abnormalities of structural layering in the neocortex and hippocampus. In contrast, conditional inactivation of the dab1 gene in the adult brain does not result in additional layering abnormalities. Furthermore, perinatal Dab1 downregulation causes behavior impairments in adult mice, such as deficits in memory, maternal care, pre-pulse inhibition, and response to cocaine. Some of these deficits were also found to be present in adolescence. We also show that D-cycloserine rescues the cognitive deficits observed in floxed dab1 mice with layering alterations in the hippocampus and neocortex. Our results indicate a causal relation between the downregulation of Dab1 protein levels during development and the structural and behavioral deficits associated with psychiatric diseases in the adult. [ABSTRACT FROM AUTHOR]

ATP is an electrically charged molecule that functions both in the supply of energy necessary for cellular activity and as an intercellular signaling molecule. Although controlled ATP secretion occurs via exocytosis of granules and vesicles, in some cells, and under certain conditions, other mechanisms control ATP release. Gap junctions, intercellular channels formed by connexins that link the cytoplasm of two adjacent cells, control the passage of ions and molecules up to 1 kDa. The channel is formed by two moieties called hemichannels, or connexons, and it has been suggested that these may represent an alternative pathway for ATP release. We have investigated the release of ATP through hemichannels from Xenopus oocytes that are formed by Connexin 38 (Cx38), an endogenous, specific type of connexin. These hemichannels generate an inward current that is reversibly activated by calcium-free solution and inhibited by octanol and flufenamic acid. This calcium-sensitive current depends on Cx38 expression: it is decreased in oocytes injected with an antisense oligonucleotide against Cx38 mRNA (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of these endogenous connexons also allows transfer of Lucifer Yellow. We have found that the release of ATP is coincident with the opening of hemichannels: it is calcium-sensitive, is inhibited by octanol and flufenamic acid, is inhibited in ASCx38 injected oocytes, and is increased by overexpression of Cx38. Taken together, our results suggest that ATP is released through activated hemichannels in Xenopus oocytes. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]