Divergent Cl^- and H⁺ pathways underlie transport coupling and gating in CLC exchangers and channels

The CLC family comprises H⁺-coupled exchangers and Cl^- channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown. Here, we use MD simulations, biochemical and electrophysiological measurements to identify two conserved phenylalanine residues that form an aromatic pathway whose dynamic rearrangements enable H⁺ movement outside the Cl^- pore. These residues are important for H⁺ transport and voltage-dependent gating in the CLC exchangers. The aromatic pathway residues are evolutionarily conserved in CLC channels where their electrostatic properties and conformational flexibility determine gating. We propose that Cl^- and H⁺ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters and suggest a unifying mechanism that describes the gating mechanism of both CLC subtypes.