Coexpression and Heteromerization of Two Neuronal K-Cl Cotransporter Isoforms in Neonatal Brain

The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters. The KCC2 gene codes for two isoforms, KCC2a and KCC2b, which differ in their N termini. The relative expression and cellular distribution of the two KCC2 protein isoforms are unknown. Here, we characterize an antibody against the KCC2a isoform and show that a previously described antibody against KCC2 is specific for the KCC2b isoform (Hubner, C. A., Stein, V., Hermans-Borgmeyer, I., Meyer, T., Ballanyi, K., and Jentsch, T. J. (2001) Neuron 30, 515–524). Immunostaining of dissociated hippocampal cultures confirms that both KCC2 isoforms are neuron-specific. Immunoblot analysis indicates that KCC2b is the major KCC2 isoform in the adult brain, whereas in the neonatal mouse central nervous system, half of total KCC2 protein is KCC2a. At this stage, the two KCC2 isoforms are largely colocalized and show similar patterns of distribution in the brain. When coexpressed in HEK293 cells, KCC2a and KCC2b proteins form heteromeric complexes. Moreover, the two isoforms can be coimmunoprecipitated from the neonatal brain, suggesting the presence of endogenous KCC2a-KCC2b heteromers. Consistent with this, native gel analysis shows that a substantial part of endogenous KCC2 isoforms in the neonatal brain constitute dimers.The neuron-specific K⁺-Cl^- cotransporter KCC2 extrudes potassium and chloride ions from neurons, thus maintaining the low intracellular chloride concentration [Cl]_i necessary for the fast hyperpolarizing actions of inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine (1). KCC2 is an ∼140-kDa plasma membrane protein that belongs to the cation chloride cotransporter (CCC)⁴ family (2, 3). CCCs, including KCC2, are thought to exist in the plasma membrane as functional oligomers, although the mechanisms whereby oligomerization affects their transport activity are unclear (4–8).We have recently shown that the KCC2 gene (alias Slc12a5) generates two mRNAs, KCC2a and KCC2b, by an alternative promoter and first exon usage (9). The difference between the KCC2a and KCC2b proteins lies in the most N-terminal part; the 40 unique amino acids in KCC2a include a putative binding sequence for the Ste20-related proline-alanine-rich kinase (SPAK). KCC2 null mutant mice deficient for both KCC2 isoforms show a disrupted breathing rhythm and die immediately after birth (10, 11), whereas selective KCC2b isoform knock-out mice exhibit spontaneous seizures but can survive up to 3 weeks after birth (9, 12). Thus, KCC2a obviously supports some vital functions of lower brain structures.In general, KCC2 expression in the CNS follows neuronal maturation; it is first detected in the postmitotic neurons of the spinal cord and brainstem and is then gradually increased in higher brain structures (13, 14). Our previous work has shown that during postnatal development, KCC2a mRNA expression remains relatively constant, whereas KCC2b mRNA is strongly up-regulated in the cortex during postnatal development (9). This indicates that KCC2b is responsible for the developmental shift from depolarizing to hyperpolarizing GABAergic responses.Here, we study the relative expression and cellular distribution of KCC2a and KCC2b proteins in the mouse brain. We characterize a new antibody specific for the KCC2a isoform and demonstrate that a previously described antibody against KCC2 (11) is specific for the KCC2b isoform. The relative expression of the KCC2 protein isoforms determined by immunoblot analysis correlates well with their mRNA levels (9). KCC2a and KCC2b proteins have a similar level and pattern of expression in the neonatal mouse brain and are colocalized in most neurons in non-cortical lower brain areas. Coimmunoprecipitation (coIP) experiments and coexpression followed by native gel analysis indicate that KCC2a-KCC2b heteromers can form in vitro and may exist in vivo.