Reconstitution of functional voltage-gated chloride channels from complementary fragments of CLC-1.
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 Leu720 abolished function. Currents were restored by coexpressing this membrane-embedded part with the lacking cytoplasmic fragment that contains domain D13, the second of the two conserved cystathionine beta-synthase (CBS) motifs present in all eukaryotic CLC proteins. However, if the cut was after Gln597 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 NH2-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.
Schmidt-Rose
T
T
Center for Molecular Neurobiology Hamburg (ZMNH), Hamburg University, Martinistrasse 52, D-20246 Hamburg, Germany.
Jentsch
T J
TJ
D000818:Animals; D018118:Chloride Channels / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating; D013329:Structure-Activity Relationship; D014981:Xenopus
D018118:Chloride Channels