APPS November 2002 Meeting Abstract 1329

Truncations and point mutations in the carboxyl tail of the skeletal muscle chloride channel, ClCN1, are known to be associated with myotonic muscle disease (eg, Q658X, A659V, R669C, F708L, E717X, Q807X, G859D, R894X and P932L)¹ and many of these ClC-1 mutants have been shown to be non functional when expressed in heterologous systems. Functionally significant domains in the tail are, however, still largely unknown and recent crystallisation of two bacterial ClC isoforms²has not helped our understanding, because, although able to catalyse a transmembrane chloride flux, they lack most of the tail. One thing that is clear is that all eukaryotic ClCs normally have two cystathionine β synthase (CBS) domains, one located near the N terminal end of the tail and the other, in ClC-1, about two thirds of the way along it. Both ClC-0 and ClC-1 have previously been functionally reconstituted from complementary pairs of co-expressed ClC-1 mRNA, where the split is between the first CBS domain (CBS1) and the second (CBS2), whereas similar attempts to restore function in splits immediately before or within CBS1 were unsuccessful. We have attempted reconstitution from several truncated transmembrane constructs of ClC-1 (ranging between Y601X and N879X, all of which were non functional when expressed alone), by co-expressing them with a variety of carboxyl tail fragments. Of particular interest were G721X co-expressed with its 721 to C terminal complement (K722-C) and G721X co-expressed with L863-C both of which gave currents indistinguishable from wild type (WT) even though the latter was missing 142 amino acids which included most of CBS2. We then asked whether all of the remaining tail was essential for function since we had previously shown that truncations of up to 100 amino acids from the full length ClC-1 had only minor effects. Even G721X co-expressed with L863-N889X was functional.