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      A Chloride Channel at the Basolateral Membrane of the Distal-convoluted Tubule : a Candidate ClC-K Channel

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          Abstract

          The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na +-Cl cotransporter located at the apical membrane, and Na +, K + ATPase at the basolateral side. Cl transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ∼9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NP o in cell-attached patches was not modified when tubules were preincubated in the presence of 10 −5 M forskolin, but the channel was inhibited by phorbol ester (10 −6 M). In addition, NP o was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM ( NP o increased threefold), or pH increased from 6.4 to 8.0 ( NP o increased 15-fold). Selectivity experiments conducted on inside-out patches showed that the Na + to Cl relative permeability was 0.09, and the anion selectivity sequence Cl ∼ I > Br ∼ NO 3 > F . Intracellular NPPB (10 −4 M) and DPC (10 −3 M) blocked the channel by 65% and 80%, respectively. The channel was inhibited at acid intracellular pH, but intracellular ATP and PKA had no effect. ClC-K Cl channels are characterized by their sensitivity to the external calcium and to pH. Since immunohistochemical data indicates that ClC-K2, and perhaps ClC-K1, are present on the DCT basolateral membrane, we suggest that the channel detected in this study may belong to this subfamily of the ClC channel family.

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          Most cited references25

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          Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

          1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.
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            Liquid junction potentials and small cell effects in patch-clamp analysis.

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              Mutations in the chloride channel gene CLCNKB as a cause of classic Bartter syndrome.

              ABSTRACT.: Inherited hypokalemic renal tubulopathies are differentiated into at least three clinical subtypes: (1) the Gitelman variant of Bartter syndrome (GS); (2) hyperprostaglandin E syndrome, the antenatal variant of Bartter syndrome (HPS/aBS); and (3) the classic Bartter syndrome (cBS). Hypokalemic metabolic alkalosis and renal salt wasting are the common characteristics of all three subtypes. Hypocalciuria and hypomagnesemia are specific clinical features of Gitelman syndrome, while HPS/aBS is a life-threatening disorder of the newborn with polyhydramnios, premature delivery, hyposthenuria, and nephrocalcinosis. The Gitelman variant is uniformly caused by mutations in the gene for the thiazide-sensitive NaCl-cotransporter NCCT (SLC12A3) of the distal tubule, while HPS/aBS is caused by mutations in the gene for either the furosemide-sensitive NaK-2Cl-cotransporter NKCC2 (SLC12A1) or the inwardly rectifying potassium channel ROMK (KCNJ1). Recently, mutations in a basolateral chloride channel CLC-Kb (CLCNKB) have been described in a subset of patients with a Bartter-like phenotype typically lacking nephrocalcinosis. In this study, the screening for CLCNKB mutations showed 20 different mutations in the affected children from 30 families. The clinical characterization revealed a highly variable phenotype ranging from episodes of severe volume depletion and hypokalemia during the neonatal period to almost asymptomatic patients diagnosed during adolescence. This study adds 16 novel mutations to the nine already described, providing further evidence that mutations in the gene for the basolateral chloride channel CLC-Kb are the molecular basis of classic Bartter syndrome. Interestingly, the phenotype elicited by CLCNKB mutations occasionally includes HPS/aBS, as well as a Gitelman-like phenotype.
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                Author and article information

                Journal
                J Gen Physiol
                The Journal of General Physiology
                The Rockefeller University Press
                0022-1295
                1540-7748
                April 2003
                : 121
                : 4
                : 287-300
                Affiliations
                Laboratoire de Physiologie, CNRS-FRE 2468, Institut des Cordeliers, 75270 Paris, France
                Author notes

                Address correspondence to Jacques Teulon, Laboratoire de Physiologie et Génomique des Cellules Rénales, CNRS-FRE 2468, Institut des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris CEDEX 06, France. Fax: (33) 146 33 41 72; E-mail: jacques.teulon@ 123456bhdc.jussieu.fr

                Article
                200208737
                10.1085/jgp.200208737
                2217373
                12668733
                f2736591-71d4-4655-9536-6006ad5754c2
                Copyright © 2003, The Rockefeller University Press
                History
                : 25 October 2002
                : 7 February 2003
                : 27 February 2003
                Categories
                Article

                Anatomy & Physiology
                pkc,patch-clamp,clc-k,kidney,chloride channel
                Anatomy & Physiology
                pkc, patch-clamp, clc-k, kidney, chloride channel

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