A Water-Organizing Residue for H+ Access in a CLC Cl−/H+ Antiporter

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H+-gated Cl− channels and Cl−/H+ antiporters. Transmembrane H+ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl− ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl−/H+ antiport rate. The x-ray crystal structure of E202Y, the most severely affected mutant, suggests that the aromatic ring of Y202 makes cross-subunit hydrophobic interaction with an I201 sidechain from neighboring subunit, which physically blocks the interfacial pathway. This mechanism is functionally supported by the minimal effect of E202Y mutation in a monomeric variant of the transporter, which does not have such an interaction. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin.