Reconstitution of Functional Voltage-gated Chloride Channels from Complementary Fragments of CLC-1

Abstract

We investigated the effect of truncations on the human muscle chloride channel CLC-1 and studied the functional complementation from partial proteins. Almost complete deletion of the cytoplasmic amino terminus did not affect currents, but truncating the intracellular COOH terminus after Leu 720 abolished function. Currents were restored by coexpressing this membraneembedded part with the lacking cytoplasmic fragment that contains domain D13, the second of the two conserved cystathionine ␤-synthase (CBS) motifs present in all eukaryotic CLC proteins. However, if the cut was after Gln 597 before the first CBS domain, no functional complementation was seen.Complementation was also obtained with channels "split" between transmembrane domains D7 and D8 or domains D8 and D9, but not when split between D10 and D11. Specificity of currents was tested by inserting point mutations in NH 2 -terminal (G188A and G230E) or COOH-terminal (K585E) fragments. In contrast to G188A and K585E, split channels did not tolerate the D136G mutation, suggesting that it may impede association from nonlinked fragments. Duplication, but not a lack of domain D8 was tolerated in "split" channels. Membrane domains D9 -D12 can insert into the membrane without adding a preceding signal peptide to ensure the extracellular amino terminus of D9. Eventually, we succeeded in reconstituting CLC-1 channels from three separate polypeptides: the amino-terminal part up to D8, D9 through CBS1, and the remainder of the cytoplasmic carboxyl terminus.In summary, several regions of CLC channels behave autonomously regarding membrane insertion and folding and mediate protein-protein interactions strong enough to yield functional channels without a direct covalent link. CLC1 chloride channels, originally identified by the expression cloning of CLC-0 from Torpedo electric organ (1), form a large gene family with at least nine members in mammals and conservation down to organisms like Escherichia coli and yeast (for review, see Ref.2). Their importance is underscored by human inherited diseases; mutations in the muscle channel CLC-1 lead to myotonia (3, 4), and those in CLC-5 lead to proteinuria and kidney stones (5).CLC proteins are structurally unrelated to other channels, including Cl Ϫ channels like ␥-aminobutyric acid and glycine receptors or the cystic fibrosis transmembrane regulator CFTR. Hydrophobicity analysis indicated 13 hydrophobic domains (D1-D13 (1)). However, newer experimental findings (6 -8) suggest the presence of only 10 (or 12) transmembrane domains ( Fig. 1, top). The topology of the D9 -D12 region, a long hydrophobic stretch interrupted only once by a short hydrophilic segment, still poses problems. Both the amino-and the carboxyl terminus reside in the cytoplasm, and the loop between D8 and D9 is glycosylated.CLC-0 forms homodimers with one pore per subunit (7, 9, 10), and this may also apply for CLC-1 (11). This one-proteinone-pore architecture distinguishes the CLC channels from voltage-gated cation channels. In shaker K channels, four hom...

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