Characterization of a Novel CLC Homolog from Citrobacter Koseri

CLC chloride-transport proteins are expressed ubiquitously and are vital to several physiological processes. This family is distinctive in that some members are chloride ion channels while others are chloride/proton antiporters. To better understand the mechanistic similarities and differences between CLC proteins, we have characterized a novel bacterial homolog from Citrobacter koseri called CLC-b. CLC-b is 24% identical and 42% similar in amino acid sequence to CLC-ec1, but lacks several amino acids near the chloride binding sites that are conserved in most CLCs. Despite lacking these regions, we found that CLC-b does transport chloride ions. CLC-b contains an isoleucine at the position equivalent to the intracellular proton transfer glutamate (E203 in CLC-ec1). Since all known CLC ion channels contain a hydrophobic residue at this position whereas all antiporters contain a protonatable residue at this position, we had hypothesized that CLC-b would be an ion channel. To our surprise, we found that CLC-b is a chloride/proton antiporter. Hence, a protonatable residue at the intracellular glutamate position is not necessary for proton transfer. We are currently working to test the ion selectivity and the effects of introducing a glutamate at this position.