ANALYSIS OF A PROTEIN REGION INVOLVED IN PERMEATION AND GATING OF THEVOLTAGE-GATED TORPEDO CHLORIDE CHANNEL CLC-0

1. The chloride channel from the Torpedo electric organ, ClC-0, is con trolled by two distinct ('fast' and 'slow') voltage-dependent gates. H ere we investigate the effects of mutations in a region after putative transmembrane domain D12. A mutation in this region has previously be en shown to change fast gating and permeation. 2. We used a combinatio n of site-directed mutagenesis with two-electrode voltage-clamp and pa tch-clamp measurements. 3. Most conservative substitutions have minor effects, while more drastic mutations change kinetics and voltage depe ndence of fast gating, as well as ion selectivity and rectification. 4 . While ClC-0 wild-type (WT) channels deactivate fully in two-electrod e voltage clamp at negative voltages, channels do not close completely in patch-clamp experiments. Open probability is increased by intracel lular chloride in a concentration- but not voltage-dependent manner. 5 . In several mutants, including K519R, the minimal macroscopic open pr obability of fast gating is larger than in WT. Mutant channels fluctua te at negative potentials between open and closed conformations. Open probability is much more effectively increased by intracellular chlori de than in WT. The observations support the idea that permeating ions inside the pore stabilize the open state. 6. Besides effects on permea tion and gating of single protopores, some mutations affect 'slow' gat ing. In summary, the region after D12 participates in fast as well as in slow gating; mutations additionally influence permeation properties .