1229 Research Article Introduction The nine members of the mammalian ClC family of Cl – channels and transporters can be divided into three subfamilies based on functional properties, sequence similarity and subcellular localization. ClC-1, ClC-2 and ClC-Ka/b, members of the most studied subfamily, share 54% sequence identity and are Cl – channels that are predominately expressed in the plasma membrane (Jentsch et al., 1999). The subfamily of Cl – /H + transporters, ClC-3, ClC-4 and ClC-5, share ~78% of their sequence and are primarily expressed in endosomal membranes (Picollo and Pusch, 2005; Scheel et al., 2005). The third subfamily, encompassing ClC-6 and ClC-7, are probably Cl – /H + transporters that are 46% identical and are predominately expressed in late endosomal and lysosomal membranes, respectively (Graves et al., 2008; Ignoul et al., 2007). ClC-7 is also found in the plasma membrane of the extracellular lysosome of the osteoclast (Kornak et al., 2001). The physiological significance of two out of the three members of the second subfamily, ClC-3 and ClC-5, have been highlighted through the discovery of mutations within the genes that encode them that lead to human disease or through the study of ClC-specific knockout mice. Mutations in the gene encoding ClC-5 (CLCN5) lead to Dent’s Disease, a renal disease characterized by proteinuria and thus implying a role for ClC-5 in re-absoprtion of proteins within the proximal tubule. This role was subsequently confirmed in the studies of Clcn5 knockout (KO) mice (Piwon et al., 2000). Although mutations in humans have yet to be found for ClC-3, studies on Clcn3 KO mice have been pivotal in identifying the functional role of this ClC family member. For example, the study of the Clcn3 KO mouse have identified a role for ClC-3 in synaptic vesicle acidification, a process that may be crucial for retinal and hippocampal health, at least in the mouse (Stobrawa et al., 2001). Although subsequent human disease and studies on knockout mice have been pivotal to the understanding of the physiological role of ClC-3 and ClC-5, mutations within the CLCN4 gene have yet to be found in humans and a study on the Clcn4 KO mice revealed no overt phenotype, thus leaving the physiological role of ClC-4 unclear (Jentsch, 2008). As ClC-4 shares a high sequence conservation of 78% with ClC- 5, a transporter known to be involved in endosomal acidification and trafficking in the proximal tubule, it has been proposed that ClC-4 may play similar roles within the kidney (Gunther et al., 2003; Hara-Chikuma et al., 2005a; Mohammad-Panah et al., 2003). Colocalization of ClC-4 with an endosomal marker, FITC-dextran, in tissue sections of the rat proximal tubule, and co-localization of endogenous ClC-4 to Rab-5a-positive endosomes of Cos7 cells indicated that ClC-4 was appropriately localized subcellularly to modulate renal endosomes directly (Mohammad-Panah et al., 2003). In support of this finding, antisense experiments in LLPKC cells that reduced expression levels of endogenous ClC-4 revealed endosomal alkalinzation and impaired endocytic uptake of ClC-4 is closely related to ClC-5, a member of the ClC family of transporters and channels. Unlike ClC-5, for which a role in the regulation of endosomal function was well established, the cellular function of ClC-4 was uncertain. In the present study, we tested for a specific role for ClC-4 in recycling endosomes by comparing transferrin (Tfn) receptor function in primary cell lines generated from ClC-4-null mice and their wild-type siblings. We found that endosomal pH is relatively alkaline and receptor-mediated uptake of Tfn is reduced in ClC-4-null fibroblasts. Surprisingly, this reduction in Tfn uptake occurs, despite a minor increase in the total surface expression of the Tfn receptor in ClC-4-null fibroblasts. As impaired Tfn uptake by ClC-4-null fibroblasts could be rescued to wild-type levels by addition of the iron chelator: desoxiferramine, the primary defect in these cells is related to the failure of iron to dissociate from Tfn, a pH-dependent event in endosomes that precedes the dissociation of Tfn from its receptor at the cell surface. Interestingly, ClC-4 depletion had no effect on epidermal growth factor receptor (EGFR) trafficking to lysosomes for degradation pointing to its specific role in recycling endosomes. These observations provide direct evidence supporting an essential role for ClC-4 in the modulation of Tfn receptor accessibility at the cell surface through its role in endosomal acidification. Key words: ClC family, Endosomal pH, Primary fibroblast cell line Summary An essential role for ClC-4 in transferrin receptor function revealed in studies of fibroblasts derived from Clcn4-null mice Raha Mohammad-Panah 1, *, Leigh Wellhauser 1,2, *, Benjamin E. Steinberg 3 , Yanchun Wang 1 , Ling Jun Huan 1 , Xiang-Dong Liu 4 and Christine E. Bear 1,2,‡ 1 Programme in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, Canada 2 Department of Biochemistry, Faculty of Medicine, University of Toronto, Canada 3 Programme in Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, Canada 4 Department of Genetics, Hospital for Sick Children, 555 University Avenue, Toronto, Canada *These authors contributed equally to this work ‡ Author for correspondence (e-mail: bear@sickkids.on.ca) Accepted 1 December 2008 Journal of Cell Science 122, 1229-1237 Published by The Company of Biologists 2009 doi:10.1242/jcs.037317 Journal of Cell Science