The nine-member CLC gene family of Cl⁻ chloride-transporting membrane proteins is divided into plasma membrane-localized Cl⁻ channels and endo-/lysosomal Cl⁻/H⁺ antiporters. Accessory proteins have been identified for ClC-K and ClC-2 channels and for the lysosomal ClC-7, but not the other CLCs. Here, we identified TMEM9 Domain Family Member B (TMEM9B), a single-span type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Co-expression of TMEM9B with ClC-3 or ClC-4 dramatically reduced transporter activity in Xenopus oocytes and transfected HEK cells. For ClC-3, TMEM9B also induced a slow component in the kinetics of the activation time course, suggesting direct interaction. Currents mediated by ClC-7 were hardly affected by TMEM9B, and ClC-1 currents were only slightly reduced, demonstrating specific interaction with ClC-3 and ClC-4. We obtained strong evidence for direct interaction by detecting significant Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with ClC-1 or ClC-7. The discovery of TMEM9B as a novel interaction partner of ClC-3 and ClC-4 might have important implications for the physiological role of these transporters in neuronal endosomal homeostasis and for a better understanding of the pathological mechanisms in CLCN3- and CLCN4-related pathological conditions.

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.

Endosomal ClC-3 and ClC-4 Cl⁻/H⁺ antiporters are essential regulators of vesicular ion homeostasis and neuronal function, yet the mechanisms controlling their activity remain incompletely understood. TMEM9A has recently emerged as an accessory β-subunit of endosomal CLC transporters. Here, we define the functional and molecular specificity of TMEM9A across the CLC family. Electrophysiological recordings in Xenopus laevis oocytes and HEK293 cells demonstrated that TMEM9A strongly suppresses ClC-3- and ClC-4-mediated transport, while producing no detectable inhibition of ClC-2 or ClC-7 and only a modest effect on ClC-1. Consistent with these functional effects, colocalization microscopy and FLIM-FRET revealed close molecular association of TMEM9A with ClC-3 and ClC-4, with FRET efficiencies of 0.19 ± 0.02 and 0.15 ± 0.02, respectively, whereas substantially weaker interactions were detected with ClC-1 and ClC-7. In the mouse brain, Tmem9a, Tmem9b, Clcn3, and Clcn4 displayed broad and overlapping expression across cortex, hippocampus, and cerebellum. Moreover, immunofluorescence analysis showed moderate colocalization of TMEM9A with ClC-3 in primary neurons and substantial colocalization with ClC-3 and ClC-4 in heterologous cells. Together, these findings establish a preferential functional and molecular association of TMEM9A with ClC-3 and ClC-4, closely mirroring the selectivity of TMEM9B, and support the TMEM9 proteins as a regulatory β-subunit system for the neuronal endosomal CLC transporters.