Variants in the CLCN3 and CLCN4 genes, encoding the neuronal endosomal Cl − /H + antiporters ClC-3 and ClC-4, respectively, are associated with neurodevelopmental disorders exhibiting broad phenotypic heterogeneity. To date, over sixty CLCN4 variants have been functionally characterized, displaying either gain- or loss-of-function effects. While ClC-3 can function independently as a homodimer, ClC-4 requires heterodimerization with ClC-3 for efficient trafficking to endosomes. CLCN4 is located on the X chromosome. Complete loss-of-function variants, such as early stop codons, are typically non-syndromic in hemizygous males and asymptomatic in heterozygous females. In contrast, certain missense variants leading to partial or complete loss of function can result in severe syndromic phenotypes in both sexes. Here, we report dominant effects of three CLCN4 variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes. To further investigate the underlying pathogenic mechanisms, we performed whole-cell patch-clamp recordings in transfected mammalian cells utilizing a bicistronic construct encoding ClC-3 and ClC-4, separated by an internal ribosomal entry site (IRES), ensuring a defined stoichiometry of co-expression. Our findings provide the first experimental evidence of dominant-negative effects of CLCN4 variants within ClC-3/ClC-4 complexes as a molecular mechanism contributing to disease. Additionally, we present a robust platform for the functional assessment of further disease-associated variants, thereby facilitating future mechanistic studies and potential therapeutic development.
Tettey‐Matey et al. (Sun,) studied this question.