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.
Uvarov
Pavel
P
Neuroscience Center, University of Helsinki, Viikinkaari 4, 00014 Helsinki, Finland. Electronic address: pavel.uvarov@helsinki.fi.
Ludwig
Anastasia
A
Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
Markkanen
Marika
M
Neuroscience Center, University of Helsinki, Viikinkaari 4, 00014 Helsinki, Finland.
Soni
Shetal
S
Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
Hübner
Christian A
CA
Department of Clinical Chemistry, Friedrich-Schiller-Universität, D-07747 Jena, Germany.
Rivera
Claudio
C
Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00014 Helsinki, Finland.
Airaksinen
Matti S
MS
Neuroscience Center, University of Helsinki, Viikinkaari 4, 00014 Helsinki, Finland. Electronic address: matti.airaksinen@helsinki.fi.
D000818:Animals; D002460:Cell Line; D019281:Dimerization; D005786:Gene Expression Regulation / Q000502:physiology*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D018377:Neurotransmitter Agents / Q000378:metabolism; D020033:Protein Isoforms / Q000096:biosynthesis; D027981:Symporters / Q000096:biosynthesis*; D000096922:K Cl- Cotransporters
D018377:Neurotransmitter Agents; D020033:Protein Isoforms; D027981:Symporters; D000096922:K Cl- Cotransporters