The human ClC chloride channel isoforms can be classified in two groups: the plasma membrane isoforms ClC-1, ClC-2, ClC-Ka and ClC-Kb, and the intracellular isoforms ClC-3, ClC-5 and ClC-7. ClC-3 and ClC-5 are localised in endosomes, where they contribute to the anion shunt current facilitating acidification of these compartments by the proton pump (Jentsch, T.J. et al. 2002). Little is known about the subcellular localisation of ClC-4. However, since it shares high sequence identity with ClC-3 and ClC-5, we investigated whether ClC-4 is localised in an intracellular compartment.

We transiently transfected ClC-4 conjugated C-terminally with GFP (ClC-4-GFP) in HEK293 cells. Expression of ClC-4-GFP in these cells was predominantly found in a dense intracellular network of membranes identified as the endoplasmic reticulum (ER) by co-staining for the sarco/endoplasmic reticulum ATPase (SERCA).

To identify the motif responsible for ER localisation of ClC-4 we generated chimaeras by exchanging segments of ClC-4 with the corresponding regions of ClC-3, and the unrelated type II plasma membrane protein Ly49E. Replacement of the C-terminal tail of ClC-3 with the corresponding sequence of ClC-4 did not change its vesicular localisation, suggesting that the putative ER retrieval motifs in the cytoplasmic C-terminus of ClC-4 are not responsible for its ER localisation. In contrast, a chimaera in which the first 71 N-terminal amino acids of ClC-3 were replaced with the corresponding segment of ClC-4 showed a localisation similar to ClC-4, as confirmed by co-localisation with SERCA. To exclude that the ER localisation of this chimaera was the consequence of ER retention due to misfolding, instead of being due to a genuine ER localisation signal, we created a mirror-image chimaera where the first 71 N-terminal amino acids of ClC-4 were substituted with the equivalent segment of ClC-3. Indeed, this chimaera showed a vesicular distribution pattern, suggesting that the N-terminus of ClC-4 contains a genuine ER localisation signal. This was confirmed by the observation that substitution of the cytoplasmic N-terminus of the plasma membrane protein Ly49E with the first N-terminal 63 amino acids of ClC-4 redistributed a large amount of the expressed protein from the plasma membrane to the ER. Moreover, no alteration of the ER localisation was found, when ClC-4 was truncated at amino acid 112 after the first transmembrane spanning domain.

Taken together, our results indicate that the first 63 N-terminal amino acids of ClC-4 contain a novel ER targeting signal that is both necessary and sufficient for localisation in the ER.