Specific Interaction of TMEM9A with Neural Endosomal Antiporters ClC-3 and ClC-4
- 1Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy
- 2Current addresses: DISTAV, Dipartimento di Scienze della Terra, dell’Ambiente e della Vita, Università di Genova,16132 Genova, Italy
- 3DIFI Lab, Dipartimento di Fisica, Università di Genova, 16146 Genova, Italy
Abstract
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.
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BibTeX
@article{rubino2026specific,
title = {{Specific Interaction of TMEM9A with Neural Endosomal Antiporters ClC-3 and ClC-4}},
author = {Eugenia Rubino and Abraham Tettey-Matey and Alice Giusto and Francesca Sbrana and Margherita Festa and Maria Antonietta Coppola and Cristiana Picco and Paola Gavazzo and Elena Angeli and Michael Pusch and Raffaella Barbieri},
journal = {Cells},
year = {2026},
month = oct,
doi = {10.3390/cells15211932},
url = {https://www.mdpi.com/2073-4409/15/21/1932},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Specific Interaction of TMEM9A with Neural Endosomal Antiporters ClC-3 and ClC-4
AU - Eugenia Rubino
AU - Abraham Tettey-Matey
AU - Alice Giusto
AU - Francesca Sbrana
AU - Margherita Festa
AU - Maria Antonietta Coppola
AU - Cristiana Picco
AU - Paola Gavazzo
AU - Elena Angeli
AU - Michael Pusch
AU - Raffaella Barbieri
PY - 2026
DA - 2026/10/24
JO - Cells
DO - 10.3390/cells15211932
UR - https://www.mdpi.com/2073-4409/15/21/1932
AB - 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.
SN - 2073-4409
ER -