Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1

Deanne H. Hryciw; Grigori Y. Rychkov; Bernard P. Hughes; Allan H. Bretag

doi:10.1074/jbc.273.8.4304

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Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1

Journal article

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Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1

Deanne H. Hryciw, Grigori Y. Rychkov, Bernard P. Hughes and Allan H. Bretag

Journal of Biological Chemistry, Vol.273(8), pp.4304-4307

1998

DOI: https://doi.org/10.1074/jbc.273.8.4304

PMID: 9468477

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Abstract

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (DD13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and DD13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys877 to Arg894.

Details

Title: Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1
Authors: Deanne H. Hryciw - University of South Australia
Grigori Y. Rychkov - The University of Adelaide
Bernard P. Hughes - University of South Australia
Allan H. Bretag (Corresponding Author) - The University of Adelaide
Publication details: Journal of Biological Chemistry, Vol.273(8), pp.4304-4307
Publisher: Elsevier Inc.
Date published: 1998
DOI: 10.1074/jbc.273.8.4304
ISSN: 1083-351X; 0021-9258
PMID: 9468477
Grant note: This work was supported by the Neuromuscular Research Foundation of the Muscular Dystrophy Association of South Australia, the Australian Research Council, and the Research Committee of the University of South Australia.
Organisation Unit: School of Health - Biomedicine
Language: English
Record Identifier: 991186520502621
Output Type: Journal article

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