The activity of the neuron-specific K+, Cl- co-transporter 2 (KCC2) is required for hyperpolarizing action of GABA and glycine. KCC2-mediated transport therefore plays a pivotal role in neuronal inhibition. Few analyses have addressed the amino acid requirements for transport-competent conformation. KCC2 consists of 12 transmembrane domains flanked by two intracellular termini. Structural analyses of a related archaeal protein have identified an evolutionary extremely conserved beta 1 strand, which links the transmembrane domain to a C-terminal dimerization interface. Here, we focused on the sequence requirement of this linker. We mutated four highly conserved amino acids of the beta 1 strand ((673)QLLV(676)) to alanine and analyzed the functional consequences in mammalian cells. Flux measurements demonstrated that L-675A significantly reduced KCC2 transport activity by 41%, whereas the other three mutants displayed normal activity. Immunocytochemistry and cell surface labeling revealed normal trafficking of all four mutants. Altogether, our results identify L-675 as a critical residue for KCC2 transport activity. Furthermore, in view of its evolutionary conservation, the data suggest a remarkable tolerance of the KCC2 transport activity to amino acid substitutions in the beta 1 strand. (C) 2012 Elsevier Inc. All rights reserved.