The Cl^– channel, CLC-5, associated with Dent’s disease in humans consists of 746 amino-acids with intracellular N and C terminals. Two cystathionine-β-synthase (CBS) domains are present at the C-terminal and are thought to be necessary for correct trafficking of the CLC-5 protein or for correct channel function (Jentsch et al. 2002). Three natural mutations of CLC-5 (R648X, R704X and the frameshift mutation 695delCfs) give rise to Dent’s disease in humans by affecting CBS domain 2.

We used site-directed mutagenesis to prepare the R648X, R704X and 695delCfs mutants of mCLC-5 contained in the mammalian expression vector pcDNA3.1-NT-GFP. The mouse mCLC-5 is 97 % identical to the human protein at the amino acid level, with virtual identity at the C-terminal (excepting for a conservative L699M difference). We then compared the cellular distributions of N-terminal GFP-mCLC-5 mutant proteins and wild-type after transient transfection of CHO-K1 and mIMCD-3 cells and imaging of positive transfectants by confocal laser scanning microscopy. Post transfection, unfixed cells were pre-incubated with TRITC-wheat germ lectin (50 µg ml^-1) for 2 min or 1 h.

In CHO-K1 cells transient transfection of wild-type mCLC-5 is associated with the appearance of a characteristic strongly outwardly rectifying DIDS-insensitive Cl^– conductance (Sayer et al. 2001). WT-mCLC-5-NT-GFP colocalises with plasma membrane TRITC-WGA (2 min incubation) as well as being present within endosomes. The R648X, R704X and 695delCfs mutants of mCLC-5 all show an altered distribution, being absent from the plasma membrane whilst being retained at a perinuclear localisation. In mIMCD-3 cells, plasma membrane expression of WT-mCLC-5-NT-GFP is absent, expression being entirely intracellular. The R648X, R704X and 695delCfs mutants of mCLC-5 are again retained at a perinuclear localisation. Furthermore, expression of CBS mutant proteins together with treatment of mIMCD-3 cells with TRITC-WGA (1 h) shows restricted endocytic uptake of the lectin.

We conclude that CBS-2 is associated with the direction of trafficking of mCLC-5. Failure to measure Cl^– currents with CBS mutants (Lloyd et al. 1996) may be associated with a failure of protein traffic to the plasma membrane as is seen in CHO-K1 cells.

This work was supported by the NKRF.