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The Journal of Neuroscience, October 11, 2006, 26(41):10407-10419; doi:10.1523/JNEUROSCI.3257-06.2006

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Development/Plasticity/Repair
Oligomerization of KCC2 Correlates with Development of Inhibitory Neurotransmission

Peter Blaesse,¹ Isabelle Guillemin,¹ Jens Schindler,¹ Michaela Schweizer,² Eric Delpire,³ Leonard Khiroug,⁴ Eckhard Friauf,¹ and Hans Gerd Nothwang¹

¹Abteilung Tierphysiologie, Fachbereich Biologie, Technische Universität Kaiserslautern, D-67653 Kaiserslautern, Germany, ²AG Elektronenmikroskopie, Zentrum für Molekulare Neurobiologie, D-20251 Hamburg, Germany, ³Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, and ⁴Neuroscience Center, University of Helsinki, FIN-00014 Finland, Helsinki

Correspondence should be addressed to Hans Gerd Nothwang, Abteilung Tierphysiologie, Fachbereich Biologie, Technische Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany. Email: nothwang{at}rhrk.uni-kl.de

The neuron-specific K⁺–Cl^– cotransporter KCC2 extrudes Cl^– and renders GABA and glycine action hyperpolarizing. Thus, it plays a pivotal role in neuronal inhibition. Development-dependent KCC2 activation is regulated at the transcriptional level and by unknown posttranslational mechanisms. Here, we analyzed KCC2 activation at the protein level in the developing rat lateral superior olive (LSO), a prominent auditory brainstem structure. Electrophysiology demonstrated ineffective KCC2-mediated Cl^– extrusion in LSO neurons at postnatal day 3 (P3). Immunohistochemical analyses by confocal and electron microscopy revealed KCC2 signals at the plasma membrane in the somata and dendrites of both immature and mature neurons. Biochemical analysis demonstrated mature glycosylation pattern of KCC2 at both stages. Immunoblot analysis of the immature brainstem demonstrated mainly monomeric KCC2. In contrast, three KCC2 oligomers with molecular masses of 270, 400, and 500 kDa were identified in the mature brainstem. These oligomers were sensitive to sulfhydryl-reducing agents and resistant to SDS, contrary to the situation seen in the related Na⁺–(K⁺)–Cl^– cotransporter. In HEK-293 cells, coexpressed hemagglutinin-tagged KCC2 assembled with histidine-tagged KCC2, demonstrating formation of homomers. Based on these findings, we conclude that the oligomers represent KCC2 dimers, trimers, and tetramers. Finally, immunoblot analysis identified a development-dependent increase in the oligomer/monomer ratio from embryonic day 18 to P30 throughout the brain that correlates with KCC2 activation. Together, our data indicate that the developmental shift from depolarization to hyperpolarization can be determined by both increased gene expression and KCC2 oligomerization.

Key words: auditory brainstem; brain development; chloride regulation; synaptic inhibition; oligomerization; cation-chloride cotransporters

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Received Jan. 16, 2006; revised Aug. 24, 2006; accepted Aug. 24, 2006.

Correspondence should be addressed to Hans Gerd Nothwang, Abteilung Tierphysiologie, Fachbereich Biologie, Technische Universität Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany. Email: nothwang{at}rhrk.uni-kl.de

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