Proton pathways and H⁺/C1^- stoichiometry in bacterial chloride transporters

H⁺Cl^- antiport behavior has recently been observed in bacterial chloride channel homologs and eukaryotic CLC-family proteins. The detailed molecular-level mechanism driving the stoichiometric exchange is unknown. In the bacterial structure, experiments and modeling studies have identified two acidic residues, E148 and E203, as key sites along the proton pathway. The E148 residue is a major component of the fast gate, and it occupies a site crucial for both H⁺ and Cl^- transport. E203 is located on the intracellular side of the protein; it is vital for H⁺, but not Cl^-, transport. This suggests two independent ion transit pathways for H⁺ and Cl^- on the intracellular side of the transporter. Previously, we utilized a new pore-searching algorithm, TransPath, to predict Cl^- and H⁺ ion pathways in the bacterial ClC channel homolog, focusing on proton access from the extracellular solution. Here we employ the TransPath method and molecular dynamics simulations to explore H⁺ pathways linking E148 and E203 in the presence of Cl^- ions located at the experimentally observed binding sites in the pore. A conclusion is that Cl^- ions are required at both the intracellular (S_int) and central (S_cen) binding sites in order to create an electrostatically favorable H⁺ pathway linking E148 and E203; this electrostatic coupling is likely related to the observed 1H ⁺/2Cl^- stoichiometry of the antiporter. In addition, we suggest that a tyrosine residue side chain (Y445), located near the Cl ^- ion binding site at S_cen, is involved in proton transport between E148 and E203.