Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States
Torben Neelands
Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States
Alexander S Powers
Department of Chemistry, Stanford University, Stanford, United States; Department of Computer Science, Stanford University, Stanford, United States; Department of Structural Biology, Stanford University, Stanford, United States; Institute for Computational and Mathematical Engineering, Stanford University, Stanford, United States
Yan Liu
Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Stanford University, Stanford, United States
Steven D Miller
Department of Chemistry, Stanford University, Stanford, United States
Grigore D Pintilie
Department of Bioengineering and Department of Microbiology and Immunology, Stanford University, Stanford, United States
J Du Bois
Department of Chemistry, Stanford University, Stanford, United States
Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States; Department of Computer Science, Stanford University, Stanford, United States; Department of Structural Biology, Stanford University, Stanford, United States; Institute for Computational and Mathematical Engineering, Stanford University, Stanford, United States
Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Stanford University, Stanford, United States; Department of Bioengineering and Department of Microbiology and Immunology, Stanford University, Stanford, United States
CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. 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 among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the 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 conformations 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. This peptide is highly conserved among species variants of CLC-2 but is not present in 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 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.
