Oligomerization of KCC2 Correlates with Development of Inhibitory Neurotransmission

Abstract

The neuron-specific K ϩ -Cl Ϫ cotransporter KCC2 extrudes Cl Ϫ 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 Ϫ 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 ϳ270, ϳ400, and ϳ500 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 ϩ -(K ϩ )-Cl Ϫ 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.

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

“…Efficient KCC2-mediated Cl − extrusion occurs when KCC2 exists in the oligomerized state (28)(29)(30). We, therefore, tested whether Neto2 could associate with the active oligomeric fraction of KCC2.…”

A Sensitive Period of Mice Inhibitory System to Neonatal GABA Enhancement by Vigabatrin is Brain Region Dependent

“…Therefore, susceptibility is expressed in the transcriptional downregulation of KCC2 as well as its functionality, determined by association to the membrane. We examined KCC2 regulation by the state of its oligomerization, which has been previously suggested to regulate KCC2 activity (Blaesse et al, 2006). Although KCC2 protein expression has been upregulated in hippocampi and was not influenced in the cerebral cortices of GVG4-14 treated mice, examination of KCC2 oligomerization has revealed a short-term enhancement and a long-term reduction of oKCC2 due to GVG exposure in both GVG4-14 hippocampi and cerebral cortices compared with Ct4-14.…”

“…In addition, KCC2 activation is regulated by a process of oligomerization, which occurs during development (Blaesse et al, 2006). We examined the effect of GVG on the expression and oligomerization pattern of KCC2 by immunoblot analysis as an additional factor influencing the efficacy of the GABAergic inhibition.…”

“…Thus, the ability of Cl À co-transporters to influence the inhibitory function of GABA A potentials is determined by additional regulatory mechanisms of KCC2, which should be taken into consideration when determining the GABAergic molecular phenotype. Such post-translational modifications include oligomerization, membrane integration, and phosphorylation (Balakrishnan et al, 2003;Blaesse et al, 2006;Kelsch et al, 2001;Lee et al, 2007;Zhang et al, 2006). Because Cl À homeostasis is determined by both NKCC1 and KCC2, we examined the interaction between these transporters as expressed by the ratio of KCC2/NKCC1 in crude cytoplasmic fraction (S2) and in the plasmamembrane-enriched fraction (LP1).…”

Different protein profiles in inferior colliculus and cerebellum: A comparative proteomic study

“…Experiments were essentially performed as described previously (Blaesse et al, 2006) and slightly modified. Proteins were obtained from the cytosolic fraction of the IC or the cerebellum of P60 rats, and equal amounts (20 -40 g) were separated by 8 -12% SDS-PAGE.…”