Preferential association with ClC-3 permits sorting of ClC-4 into endosomal compartments

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.

Keywords: ClC-3; ClC-4; chloride transport; chloride/proton exchanger; intracellular compartments; protein sorting; protein–protein interaction; sorting signals; trafficking; transporter.

Figure 1.

Transplanting the carboxyl terminus from ClC-3a modifies intracellular localization of ClC-4. A–D, confocal images of HEK293T cells heterologously expressing ClC-4, _ClC-3ClC-4, ClC-4_ClC-3, or _ClC-3ClC-4_ClC-3 and the corresponding protein topology schematics illustrating the different protein domains that were exchanged (blue). For confocal images, cells were either cotransfected with the ER marker Bcl-2 or the lysosomal marker LAMP1 and stained with the plasma membrane marker CellMasK^TM. Scale bars = 10 μm. E, box plot of Manders coefficients of the co-localization analysis between ClC-4 chimeric proteins and the corresponding intracellular marker (ClC-4/calnexin, n = 9; ClC-4/Lamp1, n = 10; _ClC-3ClC-4/calnexin, n = 8; ClC-4_ClC-3/Lamp1, n = 9; _ClC-3ClC-4_ClC-3/Lamp1, n = 9; with n being the number of fields of view with at least three to five co-transfected cells. The MC values for ClC-4_ClC-3/Lamp1 and _ClC-3ClC-4_ClC-3/Lamp1 were significantly different from values in cells expressing ClC-4/Lamp1. F, representative whole-cell recordings from HEK293T cells expressing ClC-4 or its mutant variants. G, voltage dependence of mean current amplitudes for cells expressing WT or various mutant ClC-4 constructs obtained from recordings as shown in F (ClC-4, n = 5; _ClC-3ClC-4, n = 5; ClC-4_ClC-3, n = 5; _ClC-3ClC-4_ClC-3, n = 5). Values are given as means ± S.D., with n giving the number of cells (**, p < 0.01; ***, p < 0.001; t test; ns, not significant).

Figure 2.

Transplanting the ClC-4 amino and carboxyl termini redirects ClC-3a to the endoplasmic reticulum. A–D, confocal images of HEK293T cells heterologously expressing ClC-3, _ClC-4ClC-3, ClC-3_ClC-4, or _ClC-4ClC-3_ClC-4 and the corresponding protein topology pictures to show part of the protein sequence that was substituted (green). For confocal images, cells were either cotransfected with the ER marker calnexin or the lysosomal marker LAMP1. Scale bars = 10 μm. E, box plot of Manders coefficients of the co-localization analysis for ClC-3 chimeric proteins and corresponding intracellular marker (ClC-3a/LAMP1, n = 8; ClC-3/Calnexin, n = 7; _ClC-4ClC-3/LAMP1, n = 6; ClC-3_ClC-4/Calnexin, n = 11; _ClC-4ClC-3_ClC-4/Calnexin, n = 5), with n being the number of fields of view containing at least three to five co-transfected cells. All ClC-3a chimeric constructs show MC values that were significantly different from those measured in ClC-3a. F, representative whole-cell recordings from HEK293T cells expressing ClC-3 or its mutant variants. G, voltage dependence of mean current amplitudes for cells expressing WT or various ClC constructs obtained from recordings as shown in F (ClC-3a, n = 4; _ClC-4ClC-3, n = 8; ClC-3_ClC-4, n = 4; _ClC-4ClC-3_ClC-4, n = 6). Values are given as means ± S.D., with n giving the number of cells. ***, p < 0.001, t test.

Figure 3.

The endoplasmic reticulum retention signal of ClC-4 is located in the interlinker between CBS1 and CBS2. A–F, confocal images of HEK293T cells heterologously expressing ClC-3a or ClC-4 chimeric constructs: ClC-3_{ClC-4(586–611aa)}, ClC-3_{ClC-4 (CBS1-Linker)}, or ClC-3_{ClC-4(CBS2–760aa)} (A–C) or ClC-4_ClC-3(CBS1), ClC-4_{ClC-3(CBS1-Linker)}, or ClC-4_{ClC-3(Linker)} (D–F). In the protein topology image, exchanged protein sequences are highlighted in green or blue. Cells were either co-transfected with the ER markers calnexin/Bcl-2 or the lysosomal marker LAMP1. Scale bars = 10 μm. G, box plot of Manders coefficients of the co-localization analysis for ClC-3a or ClC-4 chimeric proteins with the corresponding intracellular marker (ClC-3_{ClC-4(586–611aa)}/LAMP1, n = 9; ClC-3_{ClC-4(CBS1-Linker)}/calnexin, n = 8; ClC-3_{ClC-4(CBS2–760aa)}/LAMP1, n = 11; or ClC-4_ClC-3(CBS1)/calnexin, n = 10; ClC-4_{ClC-3(CBS1-Linker)}/calnexin, n = 7; or ClC-4_{ClC-3(CBS1-Linker)}/CellMask, n = 7; ClC-4_{ClC-3(Linker)}/calnexin, n = 13; or ClC-4_{ClC-3(Linker)}/CellMask, n = 13), with n being the number of fields of view containing at least three to five co-transfected cells. Dashed lines correspond to the average values obtained for ClC-3a/LAMP1 or ClC-3a/calnexin (left panel) and ClC-4/calnexin (right panel). MC values from ClC-3_{ClC-4(586–611aa)}/LAMP1 and ClC-3_{ClC-4(CBS2–760aa)}/LAMP1 were not significantly different from the control ClC-3a/LAMP1 (t test, p = 0.48). In contrast, ClC-3_{ClC-4(CBS1-Linker)} exhibits significantly higher MCs with calnexin than ClC-3a/calnexin (Mann-Whitney rank-sum test, p ≤ 0.001). ClC-4_{ClC-3(CBS1-Linker)} and ClC-4_{ClC-3(CBS1-Linker)} show significantly lower MCs than ClC-4/calnexin (***, p < 0.001; t test; ns, not significant).

Figure 4.

ClC-4 is targeted to distinct endosomal compartments when co-transfected with interacting CLC transporters. A–D, confocal images of HEK293T cells co-expressing ClC-4 and ClC-3a (A), ClC-3b (B), ClC-3c (C), or ClC-6 (D). Scale bars = 10 μm. E, box plot of Manders coefficients of the co-localization analysis for ClC-4 and various other CLC transporters (ClC-4/ClC-3a, n = 8; ClC-4/ClC-3b, n = 9; ClC-4/ClC-3c, n = 12; ClC-4/ClC-6, n = 9), with n being the number of fields of view containing at least three to five co-transfected cells. Co-localization analysis shows that cells co-expressing ClC-4 and different ClC-3 splice variants exhibit similar MC values but significantly larger coefficients than cells co-expressing ClC-4 and ClC-6. ***, p < 0.001; t test).

Figure 5.

Different subcellular ClC-4 distributions in WT and Clcn3^−/− astrocytes. A and B, confocal images of astrocytes from WT (A, scale bar = 10 μm) or Clcn3^−/− (B, scale bar = 30 μm) mice co-transduced with fluorescent ClC-4 fusion proteins and the late endosomal/lysosomal marker Rab7 via lentiviral gene transfer. C, box plot of Manders coefficients of the co-localization analysis for ClC-4 and Rab7 in WT or Clcn3^−/− astrocytes. MC values are significantly different for WT and Clcn3^−/− astrocytes (p < 0.001; t test). D, box plot of mean whole-cell fluorescence intensity of WT (n = 19) or Clcn3^−/− (n = 7) cells expressing ClC-4 fusion proteins (n, number of analyzed fields of view containing at least three to five cells; AU, arbitrary unit; ns, not significant).

Figure 6.

hrCNE demonstrates different stabilities of ClC-1, ClC-3, and ClC-4 homodimers. A, SDS-PAGE analysis of fluorophore-tagged proteins heterologously expressed in HEK 293T cells. B, hrCNE analysis of the same proteins. Fluorophore-tagged proteins were visualized by fluorescence scanning of the gels, respectively. C, box plot of the normalized fluorescence intensities of the protein bands assigned to homodimeric or heterodimeric bands. Normalization was performed as described under “Experimental Procedures.” MBP-ClC3b-eGFP, His-MBP-ClC-3b-eGFP N880/883Q; ClC4-eGFP, His-ClC-4-eGFP N428/431Qn; ClC4-mCherry, His-ClC-4-mChery N428/431Q. Data were obtained from seven independent experiments. p ≤ 0.001, Mann-Whitney rank-sum test.