Specific Interaction of TMEM9A with Neural Endosomal Antiporters ClC-3 and ClC-4

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.