Cryo-EM structures of the full-length human KCC2 and KCC3 cation-chloride cotransporters

The electroneutral K + -Cl − cotransporters (KCCs) play fundamental roles in trans-epithelial transport, cell volume regulation, and neuronal excitability. 1 The neuronal-specific KCC2 (SLC12A5) and glial-expressed KCC3 (SLC12A6) are essential for normal development and function of the nervous system. Mutations in KCC2 have been linked with human idiopathic generalized epilepsy 2 and schizophrenia. 3 KCC3-dependent Cl − transport across the choroid plexus epithelia is involved in the production of cerebrospinal fluid. 4 Here, we present single particle cryo-electron microscopy (cryo-EM) structures of the full-length human KCC2 and KCC3 at overall resolution of 3.2 and 3.3 Å, respectively (Fig. 1a, b ; Supplementary information , Figs. S1 , S2 and Table S1 ). The structures of KCC2 and KCC3 are very similar to each other. For simplicity, we mainly focused on KCC3 for structural analysis. KCC3 exists as a dimer in domain-swapping conformation (Fig. 1a ). A marked difference is observed in the C-terminal domain (CTD) compared with Danio rerio Na + -K + Cl − cotransporter 1 ( Dr NKCC1), which rotates ~70° clockwise as seen from cytosolic side when transmembrane (TM) domains aligned together ( Supplementary information , Fig. S3a ). The TM domain and CTD are connected through a pair of scissor helices (Fig. 1b ), which separate the two TM domains from different protomers resulting in a unique “Central Cleft” between TM domains at the cytosolic side (Fig. 1b ). The extracellular domain (ECD) of the two protomers are placed in distance (Fig. 1b ). Both TM domains, scissor helices and CTD are involved in dimerization of the transporter. The TM domain of KCC3 is captured in an inward-facing conformation, sharing the LeuT-fold as the members of the amino acids, polyamines and organocations (APC) superfamily (Fig. 1b ), with TMs 3–5 and 8–10 forming the scaffold domain and TMs 1, 2, 6 and 7 constituting the bundle domain (Fig. 1c ). Three ion-like densities are found in the unwound region of TM1 and TM6 in KCC3 ( Supplementary information , Fig. S3b , left), to which we assigned two Cl − ions and one K + ion with the consideration of both coordination environment and structural similarity with KCC1. In the cryo-EM map of KCC2, the densities for K + and one of Cl − ions are absent. Instead there is a weaker density similar to that is observed in KCC1 purified in NaCl condition 5 ( Supplementary information , Fig. S3b , right). Amino acid substitution of tyrosine (Y407 in KCC2) to phenylalanine (F441 in KCC3) causes subtle change in the ion binding site, which might provide a possible explanation for the ion coordination differences between KCC2 and KCC3 ( Supplementary information , Fig. S3c ). Fig. 1: Cryo-EM Structure of human KCC3. a Cryo-EM map of full-length human KCC3, generated by merging maps which are refined on TM/ECD and CTD, respectively. b The structure model of human KCC3 colored by domains. Carbohydrate chains were shown in sticks. Unless otherwise indicated, the same domain color scheme is applied to all figures. c N-terminal loop locates in the cytosolic entry of the transport path of KCC3, between the bundle domain and Scaffold domain. Locations of N and C lobes of N-terminal loop are highlighted by yellow and scarlet rectangles. d N lobe of N-terminal loop binding through interaction with TM1a, and TM5. e C-lobe bound to TM8 extension and CTD. f N-terminal loop binding narrows down the inward-open entry. Permeation path calculated by HOLE. 12 Left: green dots represent the permeation path of KCC3 with N-terminal loop. Right: The pore radii along the conducting passage. The result of HOLE is based on coordinates of Y232. g DIOA locates in the central cleft. The density is well fit for DIOA. h Hydrophobic interaction from TM10, scissor helices and TM12’ stabilizes DIOA. i Hydrophilic interaction from TM10 and TM12. The interaction with R617 of the TM10–11 loop stabilizes the conformation of TM10. j Conformational change induced by DIOA binding. The alignment takes the reference of one the scissor helices as in which binds DIOA. Left panel: DIOA induce conformational change of R617 and TM10. Right panel: the rotation of TMD results in the enlarged distance between CTD and of N-terminal loop. Full size image