Intracellular localization of ClC chloride channels and their ability to form hetero-oligomers.
ClC chloride channels (ClCs) can be classified into two groups in terms of their cellular localizations: ClCs present in the plasma membranes and those residing in intracellular organelles. Members of the latter group, including ClC-3, ClC-4, ClC-5, ClC-6, and ClC-7, are often co-expressed in a variety of cell types in many organs. Although the localization of individual channels within cells has been investigated, the degree of overlap between the locations of different ClCs in the same cell has not been clarified. To address this question, different combinations of ClCs, engineered to encode specific epitope tags (FLAG or HA), were either transiently or stably transfected into HEK293 cells, and we then compared the intracellular localization of the expressed channel proteins by immunofluorescence microscopy. Immunofluorescence images of the alternatively labeled channels clearly showed significant co-localization between all pair-wise combinations of ClCs. In particular, ClC-3, ClC-4, and ClC-5 showed a high degree of co-localization. As a significant degree of co-localization between ClCs was observed, we used co-immunoprecipitation to evaluate oligomer formation, and found that each ClC tested could form homo-oligomers, and that any pair-wise combination of ClC-3, ClC-4, and ClC-5 could also form hetero-oligomers. Neither ClC-6 nor ClC-7 was co-precipitated with any other channel protein. These results suggest that within cells ClC-3, ClC-4, and ClC-5 may have combinatorial functions, whereas ClC-6 and ClC-7 are more likely to function as homo-oligomers.
Suzuki
Tatsunori
T
Department of Nephrology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo Tokyo, Japan.
Rai
Tatemitsu
T
Hayama
Atsushi
A
Sohara
Eisei
E
Suda
Shin
S
Itoh
Tomohiro
T
Sasaki
Sei
S
Uchida
Shinichi
S
D015415:Biomarkers; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D000939:Epitopes; D005455:Fluorescent Antibody Technique; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008954:Models, Biological; D009842:Oligopeptides; D015388:Organelles / Q000378:metabolism*; D010455:Peptides; D014162:Transfection
D015415:Biomarkers; D018118:Chloride Channels; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide