title: KCC2 transport activity requires the highly conserved L₆₇₅ in the C-terminal β1 strand.
abstract: 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 ((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 β1 strand.
authors.affiliation: Abteilung Neurogenetik, Institut für Biologie und Umweltwissenschaften, Carl von Ossietzky Universität Oldenburg, Oldenburg, Deutschland.
authors.forename: Annika
authors.initials: A
authors.lastname: Döding
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Hartmann
authors.forename: Timo
authors.initials: T
authors.lastname: Beyer
authors.forename: Hans Gerd
authors.initials: HG
authors.lastname: Nothwang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D019143:Evolution, Molecular; D007930:Leucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D007930:Leucine; D027981:Symporters; D000096922:K Cl- Cotransporters