Structure of the human ClC-1 chloride channel

Fig 1. Structure of human ClC-1. (a) Overall topology of ClC-1 with 17 helices (αB to αR), 2 CBS domains, and key residues pinpointed. A single monomer is displayed for clarity. Helices are labelled with white letters throughout. (b) The 3.6 Å cryo-EM map (Map 0) from pH 7.5 covering the membrane domain only (contoured at σ = 13 in Pymol). Helix A (αA) and parts of the CBS domains were not resolved in the cryo-EM density maps. (c) Alternative cryo-EM map from pH 7.5 (Map 1) with the membrane and cytoplasmic CBS domains colored in cyan and red, respectively, shown at different contour levels (σ = 15 and 22 in Pymol, respectively). The map is filtered to 5 Å, representing the local resolution of the cytoplasmic domain (see also S2–S4 and S7 Figs). (d) Overall structure (generated using Map 1) with one of the monomers in pale colors. CBS, cystathionine-β-synthase; CLC, chloride channel; cryo-EM, cryo-electron microscopy.

Fig 2. The ion-conducting pathway. Ion transport in CLC proteins depends on extra- and intracellular vestibules and a connecting pore. In CLC transporters, the pore is marked by chloride ion binding sites (sext, scen, sint; not directly observed in this work) as well as specific glutamate (GluGATE, or E232; ClC-1 numbering throughout), tyrosine (TyrC, Y578), and serine (SerC, S189) residues. Chloride conductance in voltage-dependent CLC channels such as ClC-1 may involve shuttling (i) to protonated E232-Y578 (scen) from the vestibules - directly (or through a weak sint) from the intracellular side and (ii) through K231/R421 to overcome the hydrophobic barrier (including M485) from the extracellular side. (a) Comparison of the transmembrane domains of ClC-1 (colored as in Fig 1D) and ClC-K (gray), respectively. Helices are labelled with white letters throughout. (b) Schematic overview of the chloride permeation pathway with key residues pinpointed. Labels in the parentheses refer to the corresponding helices and the αC–D loop, respectively. (c–f) Side views of the pore region of ClC-1 (panels c and e; colored as in Fig 1D) with equivalent views of ClC-K (panels d and f, shown in gray)…

Fig 3. Slow gating of ClC-1 is regulated by pH and nucleotide binding through the CBS domains. (a, b) CBS domain flexibility observed at pH 7.5 but not pH 6.2 in different cryo-EM maps. The most different ones represent Map 2 and Map 3, which were calculated from the pH 7.5 data set (panel b represents a close view; see also S7 and S8 Figs). The inset of panel a represents size-exclusion chromatography profiles of ClC-1 at pH 7.5 and 6.2. The protein peak at pH 6.2 is shifted toward a higher retention volume indicating a more compact ClC-1 (see also S9 Fig). (c) Arrangement of important structural elements in the ClC-1 structure, including αD, αF, αR, and αD−E loop as well as the linker after αR, α1, and α2 in the CBS2 domain. The colors are as in Fig 1D. The ATP molecule (black) is positioned based on the location in ClC-5 (pdb-id 2J9L) [13], and the NAD (gray) placed by exploiting the same base moiety as for ATP. Note that no nucleotide is visible in our structural data, and therefore the observed structural shifts may relate to the lower pH only (the nucleotide is placed based on structures of isolated CBS domains; see panel e). Dotted areas represent putative sites for communication…

Fig 4. Myotonia-causing mutations and the putative binding pocket of the 9-AC inhibitor. (a–d) Disease-causing and experimental missense mutations in ClC-1. Substitutions that invert (from depolarization to hyperpolarization activated) or shift the voltage dependence are shown in pink (located to the extracellular side) and blue (intracellular vestibule) or green (subunit interface), respectively. Bright colors represent disease-causing (recessive, with a stronger phenotype, but not dominant mutations are underscored), whereas experimental mutations are shown in pale colors. ClC-1 is shown in white and the Cl− vestibules in purple (calculated using HOLLOW as for Fig 2). (a) Overall view with all known disease and selected experimental mutations. We note that mutations of 5 residues that cause recessive myotonia and inward rectification are facing the extracellular vestibule; 3 located in a row on the same face of on helix B (M128, S132, D136) and 2 being the pore-constricting residues (K231, R421). Therefore, the phenotype may reflect a decreased chloride affinity of an extracellularly accessible site. (b) Close view of mutations that invert voltage dependence. Helices are labelled with…