The neuron-specific K-Cl cotransporter (KCC2) is hypothesized to function as an active Cl- extrusion pathway important in postsynaptic inhibition mediated by ligand-gated anion channels, like γ-aminobutyric acid type A (GABA(A)) and glycine receptors. To understand better the functional role of KCC2 in the nervous system, we developed polyclonal antibodies to a KCC2 fusion protein and used these antibodies to characterize and localize KCC2 in the rat cerebellum. The antibodies specifically recognized the KCC2 protein which is an ~140-kDa glycoprotein detectable only within the central nervous system. The KCC2 protein display a robust and punctate distribution in primary cultured retinal amacrine cells known to form exclusively GABA(A)ergic synapses in culture. In immunolocalization studies, KCC2 was absent from axons and glia but was highly expressed at neuronal somata and dendrites, indicating a specific postsynaptic distribution of the protein. In the granule cell layer, KCC2 exhibited a distinct colocalization with the β2/β3-subunits of the GABA(A) receptor at the plasma membrane of granule cell somata and at cerebellar glomeruli. KCC2 lightly labeled the plasma membrane of Purkinje cell somata. Within the molecular layer, KCC2 exhibited a distinctly punctate distribution along dendrites, indicating it may be highly localized at inhibitory synapses along these processes. The distinct postsynaptic localization of KCC2 and its colocalization with GABA(A) receptor in the cerebellum are consistent with the putative role of KCC2 in neuronal Cl- extrusion and postsynaptic inhibition.
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