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

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl−-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl−-permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl−-permeation pathway.

of the leak-subtracted currents at -100 mV and +80 mV.For WT initial currents, the low currents at +80 mV make this quantification challenging: for 5 out of 8 experiments on WT CLC-2, the AK-42 current at +80 mV was greater than the initial WT current at +80 mV, likely due to an increase in leak over the course of the experiment (~15 minutes of voltage pulsing).Therefore, the rectification calculated for the WT initial currents (99 ± 34, SEM n=3) has substantial uncertainty.That said, such high rectification is consistent with reports of CLC-2 rectification throughout the literature (Park et al., 1998;Arreola et al., 2002;Jentsch and Pusch, 2018).On the other hand, Delta-N currents display only mild which is straightforward to quantify given the high signal:noise (high AK-42-sensitive current relative to background), and which does not change in response to voltage pulsing.For "WT-final", the rectification ratio similarly can be accurately quantified.Thus, while the high uncertainty and variance in estimating WT initial precludes statistical comparisons, these data strongly suggest that WT CLC-2 current decreases in parallel to current run-up.Data for all experiments are summarized in Figure 6 source data 1.

Figure 2 -Figure 2 -
Figure 2 -figure supplement 1. Micrograph and 2D classes and structure validation of CLC2-TM.(A) Representative motion-corrected cryo-EM micrograph.(B) 2D class averages.(C) Gold standard FSC plots calculated in cryoSPARC.(D) Local resolution of the cryo-EM map of the CLC2-TM.(E) Model validation using Q-scores (Pintilie et al., 2020) of subunit A (left) and subunit B (right).The black line represents the expected Q-score at respective resolution based on the correlation between Q-scores and map resolution.

Figure 4 -
Figure 4 -figure supplement 3. MD analysis indicates conformational flexibility of the CTD.(A) The CTD is highly mobile relative to the transmembrane domain, and often tilts upward or downward relative to its initial location.In the inset images, two representative frames from simulation are shown (orange) overlaid on the starting structure (black outline).(B) The RMSD (root mean square deviation) and RMSF (root mean square fluctuation) of the cytoplasmic domain backbone were calculated after aligning frames on the transmembrane domains.Bars show the mean of five independent simulations, each 2.0 μs in length.Error bars are 68% confidence intervals of the mean.

Figure 7 -
Figure 7 -figure supplement 1. cryoEM workflow of the CLC2-TM-AK42 single-particle cryo-EM data processing.A total of 14,300 movie stacks were collected on a 300 kV Titan Krios cryoelectron microscope.cryoSPRAC was used for 2D classification and CLC2-TM-AK42 density map was obtained after 2D classification.Relion was used for 3D classification.

Figure 7 -
Figure 7 -figure supplement 8. Structural comparison of CLC2-TM and CLC2-TM-AK42.Difference distance matrices comparing Cɑ residues on TM helices between CLC2-TM and CLC2-TM-AK42 in subunits A (A) or B (B).Residues in loops connecting helices, where there is low confidence in the model building (Q score lower than the expected Q score of 0.65), were omitted from the matrices.