CLC-type exchangers mediate transmembrane Cl– transport. Mutations altering their gating properties cause numerous genetic disorders. However, their transport mechanism remains poorly understood. In conventional models two gates alternatively expose substrate(s) to the intra- or extra-cellular solutions. In the CLCs, a glutamate was identified as the only gate; suggesting that they function according to a non-conventional mechanism. Here we show that transport in CLC-ec1, a prokaryotic homologue, is inhibited by crosslinks constraining movement of helix O far from the transport pathway. Crosslinked CLC-ec1 adopts a wild type-like structure, indicating stabilization of a native conformation. Movements of helix O are transduced to the ion pathway via a direct contact between its C-terminus and a tyrosine, a constitutive element of the second gate of CLC transporters. Therefore, the CLC exchangers have two gates that are coupled through conformational rearrangements outside the ion pathway.

CLC transporters catalyze transmembrane exchange of chloride ions and protons necessary for pH regulation of key physiological processes. Based on abundant structural information obtained for a bacterial CLC (CLC-ec1), the chloride pathway is defined by three anionic binding sites that span the length of the membrane. Recent results have suggested that protons might be transported by hopping through water chains between the extracellular and cytoplasmic gates in CLC-ce1. A fundamental question concerning the ClC Cl À /H þ antiporters is the mechanism of proton transport coupled to anion binding. Cl-binding facilitates functional H þ transport, while NO3-or SCN-appear to reduce or completely abolishe H þ coupling to anion movement. To investigate the coupling mechanism between anion binding to the central binding site (Scen) and water-mediated proton transport, we first applied free energy perturbation calculations to study the Scen anion selectivity. The binding free energy of NO3-and SCN-indicates that binding of these ions is unfavorable compared to Cl-binding. To compare the stability of water wires in the presence of different Scen binding anions, we employed molecular dynamics simulations starting from a snapshot including a preformed water wire structure, with Scen occupied by either Cl-, NO3-or SCN-, respectively. The average lifetime of the water wire is greatly reduced upon the binding of NO3-, while the SCNbinding breaks the wire immediately by inserting between the water molecules. Based on these results, we suggest that Scen anion selectivity affects the coupled proton transport in two ways: first, Scen selectivity determines whether the anion can bind stably; and second, whether the bind anions can support the transient water wire which is required for conduction of the protons.