PI-(3,5)P2-mediated oligomerization of the endosomal sodium/proton exchanger NHE9

Abstract

Na+/H+exchangers (NHE) are found in all cells to regulate intracellular pH, sodium levels and cell volume. The NHE isoform 9 (SLC9A9) fine-tunes endosomal pH, and its activity is linked to glioblastoma, epilepsy, autism spectrum and attention-deficit-hyperactivity disorders. Here, we report cryo-EM structures ofhorseNHE9 and a cysteine-variant at 3.6 and 3.1 Å resolution, respectively. We show how lysine residues, from a previously unresolved TM2-TM3 β-hairpin loop domain, are positioned above the dimerization interface and interact with the endosomal-specific PI-(3,5)P2lipid, together with residues located on dimer domain helices. Thermal-shift assays, solid-state membrane (SSM) electrophysiology and MD simulations, corroborates that NHE9 can specifically bind PI-(3,5)P2, and that its addition stabilizes the homodimer and enhances NHE9 activity. We have further determined the cryo-EM structure ofE. coliNhaA, confirming the expected coordination of cardiolipin at the dimerization interface, solidifying the concept that Na+/H+exchanger dimerization and transporter activity can be regulated by specific lipids. Taken together, we propose that the activity of NHE9 is regulated by the PI-(3,5)P2lipid upon reaching endosomes, which we refer to as an lipid-activation-upon-arrival model.