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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., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Introduction: The aim of this study was to evaluate changes in geometry following root canal preparation using R-Motion instruments with different apical sizes and tapers., Methods: Fifty-four mesial canals of mandibular molars with single curvature of angles ranging between 20° and 30° were stratified into 3 groups according to their internal anatomy (R-Motion 25/.06, 30/.04, Reciproc Blue) (n = 18 per group). Micro-computed tomography was used to standardize the samples before instrumentation and, after instrumentation, to assess canal transportation, changes in canal volume and centering ability. Canals were irrigated with 17% EDTA and sodium hypochlorite, and the final rinse included subsonic agitation of these solutions. Measurements were analyzed automatically using the Dragonfly software (Come, Montreal, Canada) and were confirmed by a technician and an endodontist, based on a previously validated methodology. The results were analyzed using the Kruskal-Wallis's and Mann-Whitney's tests. The level of statistical significance was set at 5%., Results: Significant differences were found in the coronal third for canal transportation, with Reciproc Blue R25 having greater values compared with both R-Motion instruments (P < .05) and greater changes in volume when compared with R-Motion 30/.04 (P < .05)., Conclusions: R-Motion of apical size and taper 25/.06 and 30/.04 were associated with similar changes in geometry following root canal preparation in curved mesial canals of mandibular molars, whereas Reciproc Blue was associated with greater canal transportation in the coronal root third., (Copyright © 2024 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)