KCC2 transport activity requires the highly conserved L675 in the C-terminal β1 strand

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 β1 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 β1 strand (673QLLV676) to alanine and analyzed the functional consequences in mammalian cells. Flux measurements demonstrated that L675A 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 L675 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 β1 strand.

► Investigation of the evolutionary highly conserved C-terminal β1 strand of KCC2. ► Flux measurements revealed importance of L675 within β1 for transport activity. ► No difference in expression and surface expression of the mutant. ► Example of a mutational analysis based on structural information.