RELEVANCE OF THE D13 REGION TO THE FUNCTION OF THE SKELETAL-MUSCLE CHLORIDE CHANNEL, CLC-1

Although hydropathy analysis of the skeletal muscle chloride channel p rotein, ClC-1, initially predicted 13 potential membrane spanning doma ins (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC chan nels, is located within the extensive cytoplasmic tail that makes up t he carboxyl terminus of the protein, We have examined the effect of de leting D13 (Delta D13) and the function of the carboxyl tail by removi ng the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs we re prepared and expressed using the baculovirus Sf9 insect cell system , Patch clamp analysis of chloride currents in Sf9 cells showed that o nly relatively insubstantial changes could be attributed to the expres sed fs923X, fs895X, and Delta D13 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. wa s 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 ami no adds, but not the final 125 amino acids or more, of the carboxyl ta il. Some essential. region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys(877) to Arg(894).