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      Impaired surface membrane insertion of homo- and heterodimeric human muscle chloride channels carrying amino-terminal myotonia-causing mutations

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          Mutations in the muscle chloride channel gene ( CLCN1) cause myotonia congenita, an inherited condition characterized by muscle stiffness upon sudden forceful movement. We here studied the functional consequences of four disease-causing mutations that predict amino acid substitutions Q43R, S70L, Y137D and Q160H. Wild-type (WT) and mutant hClC-1 channels were heterologously expressed as YFP or CFP fusion protein in HEK293T cells and analyzed by whole-cell patch clamp and fluorescence recordings on individual cells. Q43R, Y137D and Q160H, but not S70L reduced macroscopic current amplitudes, but left channel gating and unitary current amplitudes unaffected. We developed a novel assay combining electrophysiological and fluorescence measurements at the single-cell level in order to measure the probability of ion channel surface membrane insertion. With the exception of S70L, all tested mutations significantly reduced the relative number of homodimeric hClC-1 channels in the surface membrane. The strongest effect was seen for Q43R that reduced the surface insertion probability by more than 99% in Q43R homodimeric channels and by 92 ± 3% in heterodimeric WT/Q43R channels compared to homodimeric WT channels. The new method offers a sensitive approach to investigate mutations that were reported to cause channelopathies, but display only minor changes in ion channel function.

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          Most cited references 60

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          Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs.

          The subunit stoichiometry of the mammalian K+ channel KV1.1 (RCK1) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit (Y379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.
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            X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity.

            The ClC chloride channels catalyse the selective flow of Cl- ions across cell membranes, thereby regulating electrical excitation in skeletal muscle and the flow of salt and water across epithelial barriers. Genetic defects in ClC Cl- channels underlie several familial muscle and kidney diseases. Here we present the X-ray structures of two prokaryotic ClC Cl- channels from Salmonella enterica serovar typhimurium and Escherichia coli at 3.0 and 3.5 A, respectively. Both structures reveal two identical pores, each pore being formed by a separate subunit contained within a homodimeric membrane protein. Individual subunits are composed of two roughly repeated halves that span the membrane with opposite orientations. This antiparallel architecture defines a selectivity filter in which a Cl- ion is stabilized by electrostatic interactions with alpha-helix dipoles and by chemical coordination with nitrogen atoms and hydroxyl groups. These findings provide a structural basis for further understanding the function of ClC Cl- channels, and establish the physical and chemical basis of their anion selectivity.
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              The influence of potassium and chloride ions on the membrane potential of single muscle fibres.

               P HOROWICZ,  A Hodgkin (1959)

                Author and article information

                [1 ]Institut für Neurophysiologie, Medizinische Hochschule Hannover , Carl-Neuberg-Straße 1, Hannover, Germany
                [2 ]Hôpital Pitié-Salpêtrière , 47–83 Boulevard de l’Hôpital,Paris, France
                [3 ]Abteilung Molekulare Pharmakologie, RWTH Aachen University Templergraben 55, Aachen, Germany
                [4 ]Institute of Complex Systems-Zelluläre Biophysik (ICS-4) , Forschungszentrum Jülich, Germany
                Author notes
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                27 October 2015
                : 5
                Copyright © 2015, Macmillan Publishers Limited

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