Conformational changes required for H(+)/Cl– exchange mediated by a CLC transporter

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.