Substrates Coupling and Inhibition of the human Na+- dependent Cl-/HCO3-exchanger

Maintaining pH homeostasis is essential for neuronal excitability and for regulating cerebrospinal fluid (CSF). The Na+-dependent Cl-/HCO3- exchanger, NCBE (SLC4A10), abundant in the choroid plexus, contributes to CSF pH control by mediating bicarbonate flux, yet its molecular mechanism has remained unclear. Here we report cryo-EM structures of human NCBE in multiple functional states, revealing conserved binding sites for Na+, Cl-, and HCO3-, as well as an unexpected K+ site indicating relaxed cation selectivity. The inhibitor DIDS binds above the substrate cavity, locking NCBE in an outward-facing conformation and defining a subfamily-selective inhibitory mechanism. Functional assays and molecular dynamics simulations validate ion coordination and inhibitor effects. Comparison of cryo-EM structures with an AlphaFold3 inward-facing model supports an elevator-like mechanism and completes the conformational cycle of Na+-coupled HCO3- transporters. Together, these findings define the structural basis of NCBE and provide a framework for understanding ion coupling and pharmacological modulation within the SLC4 family.