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Cryo-EM structures of the human cation-chloride cotransporter KCC1

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Science  25 Oct 2019:
Vol. 366, Issue 6464, pp. 505-508
DOI: 10.1126/science.aay3129
  • Fig. 1 Overall structure of human KCC1 determined in KCl and GDN.

    (A) Side view of a three-dimensional reconstruction of KCC1 with each subunit individually colored. The gray bars on either side of the structure define the position (top and bottom) of the cell membrane. (B) Cartoon representation of the KCC1 dimer in the same orientation as the electron microscopy maps in (A). N-Acetylglucosamine (NAG) and putative GDN molecules are shown as green and cyan sticks, respectively. The three ions are shown as purple (K+) and orange (Cl) spheres. N and N′ indicate the N termini; C and C′ indicate the C termini. (C) Topology and domain arrangement of the KCC1 subunit. The three ions are shown as purple (K+) and orange (Cl) circles. (D) Structure of one KCC1 subunit in intracellular (left) and side (right) views. Each domain is colored the same as in (C). Side chains of two pairs of Cys residues that form disulfide bonds are shown as yellow sticks. Numbers indicate transmembrane helices. TM1, TM2, TM6, and TM7 form the core domain; TM3 to TM5 and TM8 to TM10 form the scaffold domain.

  • Fig. 2 The ion binding sites in KCC1.

    (A) The potassium and chloride ions bind at the breakage points of TM1 and TM6 of KCC1 in KCl and GDN. Two tyrosine residues involved in the ion coordination are shown as sticks. (B and C) Magnified views of the potassium and chloride binding sites in the KCC1 structure in KCl (B) or NaCl (C) from the same orientation. In both (B) and (C), all electron densities are shown at the same contour level of 4.5σ. Numbers show distances in angstroms between the ions and coordinating atoms. (D) K+ influx in Xenopus laevis oocytes injected with wild-type (WT) and mutant mouse KCC3 complementary RNA and measured under isosmotic (basal) and hypoosmotic (stimulated) conditions. The Western blot shows that all mutants are expressed at similar levels as the wild type. The table shows equivalent residues in human KCC1 and mouse KCC3. For more sequence alignments, see fig. S1. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

  • Fig. 3 An inward-facing conformation of KCC1 in KCl and GDN.

    (A) The surface-rendered KCC1 model shows a cytosolic-facing cavity. The yellow dashed lines indicate the opening of the intracellular gate. (B) Polar residues on the surface of the cytosolic-facing cavity. (C) Hydrophobic residues form a constriction above ion binding sites. (D) The extracellular gate is occluded by extracellular linker EL6-EL7 (pink) and pre-6a loop and post-EL5 loop (orange). (E) ECD dimerization stabilizes the closed state of the extracellular gates. For clarity, TM4, TM5, TM11, and TM12 are omitted in (B) and (C), as is TM4 in (E).

  • Fig. 4 Structural comparisons of human KCC1 and DrNKCC1.

    (A) The structural superimposition of KCC1 (blue) and DrNKCC1 (green) [Protein Data Bank (PDB) 6NPL] in the TMD. For clarity, large extracellular or intracellular loops are not shown. (B) The K+ and Cl bind at similar positions in KCC1 (blue) and DrNKCC1 (green). In the KCC1 structure, K+ and Cl are shown as purple and orange spheres, respectively, whereas in DrNKCC1, all three ions are shown as green spheres. (C) Compared with DrNKCC1 (green), KCC1 (blue) loses the Na+ binding site. The Na+ in the DrNKCC1 structure is shown as a cyan sphere. Red numbers show the distances in angstroms between Cα atoms of equivalent residues in KCC1 and DrNKCC1.

Supplementary Materials

  • Cryo-EM structures of the human cation-chloride cotransporter KCC1

    Si Liu, Shenghai Chang, Binming Han, Lingyi Xu, Mingfeng Zhang, Cheng Zhao, Wei Yang, Feng Wang, Jingyuan Li, Eric Delpire, Sheng Ye, Xiao-chen Bai, Jiangtao Guo

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Supplementary Text 
    • Figs. S1 to S20
    • Table S1
    • References 

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