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

Neumann, C. M. T.; Rosenbaek, L. L.; Flygaard, R. K.; Habeck, M.; Karlsen, J. L.; Wang, Y.; Lindorff-Larsen, K.; Gad, H. H.; Hartmann, R.; Lyons, J.; Fenton, R. A.; Nissen, P.

doi:10.1101/2021.11.11.468215

bioRxiv · 10.1101/2021.11.11.468215

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

Neumann, C. M. T.·Rosenbaek, L. L.·Flygaard, R. K.·Habeck, M.·Karlsen, J. L.·Wang, Y.·Lindorff-Larsen, K.·Gad, H. H.·Hartmann, R.·Lyons, J.·Fenton, R. A.·Nissen, P.

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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 [A] 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.

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Neumann, C. M. T., Rosenbaek, L. L., Flygaard, R. K., Habeck, M., Karlsen, J. L., Wang, Y., Lindorff-Larsen, K., Gad, H. H., Hartmann, R., Lyons, J., Fenton, R. A., Nissen, P.. 2021-11-12. Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity. https://doi.org/10.1101/2021.11.11.468215

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