Dominant Action of CLCN4 Neurodevelopmental Disease Variants in Heteromeric Endosomal ClC-3/ClC-4 Transporters

Highlights: What are the main findings? Several CLCN4 variants exert dominant effects within ClC-3/ClC-4 heterodimers, as demonstrated using electrophysiological assays. The study provides the first evidence of dominant behavior of CLCN4 variants in mixed ClC-3/ClC-4 complexes and introduces a robust platform for assessing additional disease-associated variants. What are the implications of the main findings? Dominant effects of CLCN4 variants may help explain the broad phenotypic variability observed in neurodevelopmental disorders linked to CLCN4, including severe presentation in individuals with partial or complete LoF missense variants. The new experimental platform enables systematic evaluation of emerging CLCN4 and CLCN3 variants, improving genotype–phenotype interpretation and supporting future diagnostic and therapeutic strategies. Variants in CLCN3 and CLCN4, encoding the neuronal endosomal Cl−/H+ antiporters ClC-3 and ClC-4, are linked to neurodevelopmental disorders with broad phenotypic variability. Over sixty CLCN4 variants have been functionally characterized, showing gain- or loss-of-function (GoF or LoF) effects. While ClC-3 can function as a homodimer, ClC-4 depends on heterodimerization with ClC-3 for efficient endosomal trafficking. CLCN4, located on the X chromosome, exhibits diverse pathogenic outcomes: complete LoF variants often cause non-syndromic presentations in hemizygous males and are asymptomatic in heterozygous females, whereas certain missense variants with partial or complete LoF produce severe syndromic phenotypes in both sexes. Here, we demonstrate dominant effects of three CLCN4 variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes and whole-cell patch-clamp recordings in mammalian cells co-expressing both proteins via a bicistronic IRES construct. Our findings provide the first evidence of dominant-negative CLCN4 effects within ClC-3/ClC-4 complexes and establish a platform for functional analysis of additional disease-associated variants.