{"doi":"10.7554/elife.90648.2","title":"CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism","abstract":"Abstract 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.","journal":"eLife","year":2024,"id":493070,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9518,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1081345,"name":"Torben R. Neelands","orcid":null,"position":1,"is_corresponding":false},{"id":234445,"name":"Alexander S. Powers","orcid":null,"position":2,"is_corresponding":false},{"id":363323,"name":"Yan Liu","orcid":"0000-0001-5683-8695","position":3,"is_corresponding":false},{"id":1080823,"name":"S. D. Miller","orcid":"0000-0002-6502-9299","position":4,"is_corresponding":false},{"id":228227,"name":"Grigore Pintilie","orcid":"0000-0002-0848-5335","position":5,"is_corresponding":false},{"id":336181,"name":"J. Du Bois","orcid":"0000-0001-7847-1548","position":6,"is_corresponding":false},{"id":232528,"name":"Ron O. Dror","orcid":"0000-0002-6418-2793","position":7,"is_corresponding":false},{"id":91649,"name":"Wah Chiu","orcid":"0000-0002-8910-3078","position":8,"is_corresponding":false},{"id":249302,"name":"Merritt Maduke","orcid":"0000-0001-7787-306X","position":9,"is_corresponding":false},{"id":1080822,"name":"Mengyuan Xu","orcid":"0000-0002-1734-4163","position":0,"is_corresponding":true}],"reference_count":96,"raw_metadata":null,"created_at":"2026-07-19T02:09:00.148120Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}