CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Figures

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  Figure 1.. Gating conformations in CLC transporters and channels. (A) Cartoon depictions of the CLC anion-permeation pathway. The “gating glutamate” (Egate) can occupy anion binding sites within the permeation pathway (“down” and “middle” conformations, occupying the central (Scen) and external (Sext) anion-binding sites respectively) or away from these sites (“up” and “out” conformations). Coupling of Egate movement to Cl−/H+ binding/unbinding and global conformational change generates 2:1 Cl−/H+ exchange. (B) Egate is required for voltage-dependent gating in CLC channels but has been observed structurally only in CLC-1 (PDB ID: 6coy), in the “out” position. In contrast to CLC-1, which is predominantly open at zero mV, CLC-2 is predominantly closed. The open probability (Po) versus voltage (V) traces are based on published data reported for CLC-1 (Lisal and Maduke, 2008) and CLC-2 (Stolting et al., 2013).

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  Figure 2.. CryoEM structure of the human CLC-2 channel. Overall structure of the transmembrane domain (“CLC2-TM”) at 2.46 Å. The identical subunits of the homodimer are shown in purple and gray, Cl− ions are shown as green balls. (A) cryoEM density map with model overlay (contour level: 0.71, 5.5 σ) and (B) model only with dashed arrows indicating the locations of pathways detected by Caver: orange, primary Cl− pathway common to all CLCs; yellow, secondary Cl− pathway detected in CLC-2 and CLC-1. Details of the pathways are presented in Figure 3. The secondary pathway, whose function is not yet known, was so named when first observed in the CLC-1 structure (Park and MacKinnon, 2018). (C) Zoomed-in views showing cryoEM density overlay of bound Cl− and key residues – Egate (E205) and inner-gate residues SerC (S162) and TyrC (Y553). Scen is indicated with a green dashed circle. Contour level: 1.1, 7.2 σ (middle panel); 0.97, 6.3 σ (right panel).

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  Figure 3. Pore profile detected in CLC-2. (A) Left: The primary (orange) and secondary (yellow) pore detected in CLC-2 using Caver. Middle: The detected pore radii are displayed in dots as a color map. Right: Zoomed-in view of the restricted (narrowest) site of the pore profile, using the same coloring as in the middle panel. Egate, SerC and TyrC are shown as sticks. (B) Pore profile for CLC-1 (PDB ID:6coy), with display as in panel A.

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  Figure 4.. Different conformations of the CTD. (A). Overall structure of the two CLC-2 conformations. Left: CLC2-CTDsym at 2.75 Å. Right: CLC2-CTDasym at 2.76 Å. The subunits of the homodimer are shown in orange (CLC2-CTDsym) or yellow (CLC2-CTDasym) and gray (for both). The subunits adopting different CTD conformations are shown in orange and yellow, while the subunits sharing the same CTD conformation are shown in gray. The dashed line demarcates the transmembrane (TM) and cytoplasmic (CTD) domains. A hairpin structure detected at the intracellular pore is shown in blue. (B) Overlay of two conformations. (C) Hinge movement of the CTD between two conformations. The middle panel shows an overlay of CLC2-CTDsym (orange and gray) and CLC1-CTDasym (yellow and gray), with the CTDs highlighted in boxes. For the subunits that adopt different conformations (orange and yellow), the double-headed arrow indicates the change between the two conformations. The RMSD between the subunits shown in orange/yellow is 6.9 Å, while the RMSD for the subunits shown in gray is only 1.3 Å. Alignments of only the CTDs are shown at left and right; in these alignments, the low RSMD values (0.9 Å and 1.0 Å,…

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  Figure 5.. The blocking hairpin structure corresponds to an N-terminal sequence of CLC-2. (A). Left: The hairpin structure seen in the cryoEM density (blue mesh) fits N-terminal residues 14–28 (blue). Right: The subunit in CLC2-CTDasym structure that contains a CTD with rotated orientation lacks density for the hairpin structure. In this structure, the residues linking TM helices J and K are not resolved. The dark blue circles indicate this unresolved region (right panel) and the corresponding resolved J-K helix that occurs in the other subunits. (B) Model validation using Q-scores for the N-terminal hairpin: orange and light orange for subunits A & B of CLC2-CTDsymsubunit; yellow for subunit A (the hairpin-containing subunit) of CLC2-CTDasym. The black line represents the expected Q-score at 2.75 Å based on the correlation between Q-scores and map resolution. (C) Residues on the transmembrane domain interact with the N-terminal hairpin. The J-K helical linker and TM Helices C, D, R, and J are labeled with arrows. Inset: Zoomed-in view of two hydrogen bonds formed between the hairpin structure and the TM domain: Q19-Q153 and T20-R363. Mutation of these two residues (Q25 and T26 in rat…

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  Figure 6. Patch-clamp experiments support CLC-2 channel block by the N-terminal hairpin structure. (A) Representative currents from WT and Delta-N CLC-2, recorded using the whole-cell patch clamp configuration in response to the voltage protocol shown. (B) WT CLC-2 recording from panel A, shown on an expanded scale. (C) Summary of current levels measured for WT and Delta-N CLC-2 at the end of the 1-s voltage pulse to −100 mV. Data are from six independent transfection samples, in each case with WT and Delta-N recorded on the same day following transfection. WT: −600 ± 102 pA (SEM, n=8);Delta-N: −3300 ± 630 pA (SEM, n=8). (D) Western blot detection of biotinylated surface-expressed CLC-2 from three independent experiments. (E) Summary data for electrophysiology and surface-biotinylation experiments. Points representing individual experiments for Delta-N surface biotinylation (each normalized to WT) are shown. Individual data points for the electrophysiology experiments are shown in panel C. (F) Representative examples of experiments to evaluate current run-up in WT and Delta-N CLC-2. Left panels: Time course data. Following an initial voltage-family measurement (I-V protocol as in panel A,…

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  Figure 7. Bound AK-42 blocks the Cl− pathway in CLC-2. (A) TM-domain structure of the CLC-2 structure in complex with AK-42 (CLC2-TM-AK42) at 2.74 Å. The identical subunits of the homodimer are shown in hot pink and gray; Cl− ions are shown as green balls. Bound AK-42 is highlighted with a yellow circle on the pink subunit. The cyan arrow indicates the Cl− pathway. (B) Zoomed-in view of the AK-42 binding site(contour level: 0.91, 6.0 σ). (C) A sliced view of CLC-2 showing AK-42 (yellow carbon atoms; CPK coloring on non-carbon atoms) and Egate (CPK sticks). Both AK-42 and Egate obstruct the Cl− pathway, displayed as a color map.

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  FIGURE 8:. CLC-2’s gating is illustrated based on our new structural data. Opening of each protopore requires at least two events – unblock by the N-terminal hairpin (“N-ter”) and rotation of Egate away from the permeation pathway. The dashed green arrow represents the Cl− conduction pathway. Hairpin unblock (ball and chain gating) is coupled to conformational change of the CTD: rotation of the CTD towards the membrane precludes hairpin binding, thus facilitating channel opening. Opening of the channel pore following these two events is depicted for one subunit of the homodimer (filled green arrow).