| Autor: |
Festa M; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Coppola MA; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Angeli E; DIFI Lab, Dipartimento di Fisica, Università di Genova, 16146 Genova, Italy., Tettey-Matey A; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Giusto A; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Mazza I; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Gatta E; DIFI Lab, Dipartimento di Fisica, Università di Genova, 16146 Genova, Italy., Barbieri R; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Picollo A; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Gavazzo P; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Pusch M; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Picco C; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy., Sbrana F; Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genova, Italy. |
| Abstrakt: |
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. |
