Mutation of external glutamate residue reveals a new intermediate transport state and anion binding site in a CLC Cl/H antiporter.

The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl channels and Cl/H antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Glu, could explain the transport cycle of CLC-type Cl/H antiporters. However, the presence of multiple conformations (, , and ) of the Glu has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from and cmCLC from a thermophilic red alga, Thus, we aimed to investigate further the heterogeneity of Glu-conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Glu mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the "" conformation. We also found that an extra anion can be located above the external Cl-binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl-binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Glu can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.