1. Identification of three cysteines as targets for the Zn2+ blockade of the human skeletal muscle chloride channel.
- Author
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Kürz LL, Klink H, Jakob I, Kuchenbecker M, Benz S, Lehmann-Horn F, and Rüdel R
- Subjects
- Cell Line, Chloride Channels genetics, Cysteine genetics, Diethyl Pyrocarbonate pharmacology, Histidine genetics, Histidine metabolism, Humans, Hydrogen-Ion Concentration, Muscle Proteins genetics, Muscle, Skeletal cytology, Muscle, Skeletal metabolism, Mutagenesis, Site-Directed, Chloride Channels antagonists & inhibitors, Cysteine metabolism, Muscle Proteins antagonists & inhibitors, Muscle, Skeletal drug effects, Zinc pharmacology
- Abstract
Currents through the human skeletal muscle chloride channel hClC-1 can be blocked by external application of 1 mM Zn2+ or the histidine-reactive compound diethyl pyrocarbonate (DEPC). The current block by Zn2+ strongly depends on the external pH (pKa near 6.9), whereas the block by DEPC is rather independent of the pH in the range of 5.5 to 8.5. To identify the target sites of these reagents, we constructed a total of twelve cysteine- and/or histidine-replacement mutants, transfected tsA201 cells with them, and investigated the resulting whole-cell chloride currents. The majority of the mutants exhibited a similar sensitivity toward Zn2+ or DEPC as wild type (WT) channels. Block by 1 mM Zn2+ was nearly absent only with the mutant C546A. Four mutants (C242A, C254A, H180A, and H451A) were slightly less sensitive to Zn2+ than WT. Tests with double, triple, and quadruple mutants yielded that, in addition to C546, C242 and C254 are also most likely participating in Zn2+-binding.
- Published
- 1999
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