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KCC2 is one of four known isoforms of the K-Cl cotransporter with an expression pattern restricted to neurons. It mediates efflux of Cl- across neuronal membranes and plays an important role in GABAergic and glycinergic neurotransmission. To understand the molecular basis for neuronal specificity of KCC2 expression, we isolated and sequenced portions of the KCC2 gene, including some of its 5' flanking (control) region. We found a 21-bp sequence, within intron 1, that shares 80% homology to the consensus site for neuronal-restrictive silencing factor binding. We demonstrated that this specific sequence of the KCC2 gene promotes transcriptional regulation by showing that nuclear proteins isolated from a mouse neural progenitor cell line interact with this 21-bp element and by establishing that this element silences reporter gene expression in nonneuronal cells.

We examined the expression of the KCC2 isoform of the K-Cl cotransporter in the developing and adult brain, using an affinity-purified antibody directed against a unique region of the KCC2 protein. Expression was shown to be limited to neurons at the cell bodies and cell processes in the hippocampus and cerebellum. Expression seemed to be the highest at the end of processes that originated from the CA1 pyramidal cells. Developmental up-regulation of KCC2 expression was demonstrated in the entire rat brain by Northern and Western blot analyses, and in the hippocampus by immunofluorescence. Level of KCC2 expression was minimal at birth and increased significantly during postnatal development. This pattern of expression was opposite to the one of the Na-K-2Cl cotransporter that is highly expressed in immature brain and decreases during development. The up-regulation of the K-Cl cotransporter expression is consistent with the developmental down-regulation of the intracellular Cl- concentration in neurons. The level of intracellular Cl-, in turn, determines the excitatory versus inhibitory response of the neurotransmitter gamma-aminobutyric acid in the immature versus mature brain. Finally, KCC2 expression was shown in dorsal root ganglion neurons, demonstrating that expression of the cotransporter is not strictly confined to central nervous system neurons.

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