Conformational Changes Required for Chloride Ion Permeation in the CLC-ec1 Exchanger

In the classical view, channels and transporters are different in their transport rates and mechanisms. Interestingly, the CLC protein family contains both chloride ion channels and H+/Cl- exchangers. The CLC-ec1 exchanger, for which high-resolution structures and functional data are available, provides a framework to understand the mechanical features that distinguish between coupled and uncoupled transport.Although mutational studies have identified key residues regulating chloride transport and coupling to protons, the detailed permeation mechanism remain unresolved. We performed molecular dynamics simulations to better understand the structural features that regulate chloride permeation. The simulations show that the distance between residues S107 and Y445, forming the internal gate, fluctuates extensively. These fluctuations are correlated to the conformation of helix O, which can be straight or kinked as seen in the X-ray structure. Potential of mean force calculations show that, in its straight conformation, helix O limits the fluctuation of the internal gate and thus impede ion permeation. On the contrary, the kinked conformation of helix O frees Y445 thereby favoring the opening of the internal gate and the translocation of a Cl- ion between the central and internal binding sites. Breaking salt-bridges by the protonation of glutamic acid near the intracellular mouth of the pore favors fluctuations and opening of the gate.In functional experiments, constraining the movement of helix O via a crosslink to helix Q between A399 and A432 significantly reduces ion transport mediated by CLC-ec1. Our simulations show that this crosslink favors the straight conformation of helix O, thus decreasing the opening probability of the internal gate. Taken together, the molecular mechanics and functional data show that ion permeation requires opening of the internal gate controlled by the conformation of helix O.