Structural basis of ClC-3 transporter inhibition by TMEM9 and PtdIns(3,5)P2

The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters; mutations of the genes encoding these transporters are associated with numerous diseases. Despite their critical roles, the mechanisms by which CLC transporters are regulated are poorly understood. Here we show that two related accessory β-subunits, TMEM9 and TMEM9B, directly interact with ClC-3, ClC-4 and ClC-5. Cryo-electron microscopy structures reveal that TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl^- ion pathway. Unexpectedly, we find that phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) stabilizes the interaction between TMEM9 and ClC-3 and is required for proper regulation of ClC-3 by TMEM9. Collectively, our findings reveal that TMEM9 and PtdIns(3,5)P₂ collaborate to regulate endosomal ion homeostasis by modulating the activity of ClC-3.