Abstract The sodium-potassium-chloride transporter NKCC1 (SLC12A2) performs Na + -dependent Cl − and K + ion uptake across plasma membranes. NKCC1 is important for regulating e.g. cell volume, hearing, blood pressure, and chloride gradients defining GABAergic and glycinergic signaling in brain. Here, we present a 2.6 Å resolution cryo-electron microscopy (cryo-EM) structure of human NKCC1 in the substrate-loaded (Na + , K + , 2 Cl − ) and inward-facing conformation adopting an occluded state that has also been observed for the SLC6 type transporters MhsT and LeuT. Cl − binding at the Cl1 site together with the nearby K + ion provide a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl − ion binding at the Cl2 site seems to undertake a structural role similar to a conserved glutamate of SLC6 transporters and may allow for chloride-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations we describe the Na + binding site and a putative Na + release pathway along transmembrane helix 5. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity