Abstract
ClC-4 is an intracellular Cl-/H+ exchanger that is highly expressed in the brain and whose dysfunction has been linked to intellectual disability and epilepsy. Here we studied the subcellular localization of human ClC-4 in heterologous expression systems. ClC-4 is retained in the endoplasmic reticulum (ER) upon overexpression in HEK293T cells. Co-expression with distinct ClC-3 splice variants targets ClC-4 to late endosome/lysosomes (ClC-3a and ClC-3b) or recycling endosome (ClC-3c). When expressed in cultured astrocytes, ClC-4 sorted to endocytic compartments in WT cells but was retained in the ER in Clcn3-/- cells. To understand the virtual absence of ER-localized ClC-4 in WT astrocytes, we performed association studies by high-resolution clear native gel electrophoresis. Although other CLC channels and transporters form stable dimers, ClC-4 was mostly observed as monomer, with ClC-3-ClC-4 heterodimers being more stable than ClC-4 homodimers. We conclude that unique oligomerization properties of ClC-4 permit regulated targeting of ClC-4 to various endosomal compartment systems via expression of different ClC-3 splice variants.
Highlights
ClC-4 is an intracellular Cl؊/H؉ exchanger that is highly expressed in the brain and whose dysfunction has been linked to intellectual disability and epilepsy
These results indicate that ClC-4 lacks the endosomal targeting sequences of ClC-3 and that a carboxyl-terminal endoplasmic reticulum (ER) retention signal is responsible for the subcellular localization of ClC-4
ClC-3 and ClC-4 are two closely related intracellular chloride/proton exchangers that co-exist in neurons, glia, muscle, heart, and epithelial cells [3, 5, 7, 36, 37]
Summary
ClC-3 and ClC-4 are chloride/proton exchangers that reside primarily in intracellular organelles [1, 2] of the brain, heart, skeletal muscle, and epithelia [3, 4]. The functional relevance of ClC-3 and ClC-4 in the central nervous system is illustrated by pronounced hippocampal and retinal degeneration in Clcn3Ϫ/Ϫ knockout animal models [5,6,7] as well as by naturally occurring CLCN4 mutations in patients with X-linked intellectual disability or epilepsy [8, 9]. The cellular roles of ClC-3 and ClC-4 have remained insufficiently understood, mostly because of unclear subcellular localization of these two transport proteins. The subcellular localization of ClC-4 was studied in multiple preparations, with conflicting results. Okkenhaug et al [10] described endoplasmic reticulum (ER) localization of ClC-4 upon heterologous expression in mammalian cells, endogenous ClC-4 inserts into the early endosomes and into. The authors declare that they have no conflicts of interest with the contents of this article
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