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ORIGINAL RESEARCH article

Front. Mol. Neurosci.
Sec. Brain Disease Mechanisms
Volume 17 - 2024 | doi: 10.3389/fnmol.2024.1505722
This article is part of the Research Topic SLC12A5-Dependent Neurological Disorders View all 4 articles

Uncovering novel KCC2 regulatory motifs through a comprehensive transposon-based mutant library

Provisionally accepted
Pavel Uvarov Pavel Uvarov 1Satoshi Fudo Satoshi Fudo 2Cem Karakus Cem Karakus 3Andrey Golubtsov Andrey Golubtsov 1Federico Rotondo Federico Rotondo 1Tatiana Sukhanova Tatiana Sukhanova 1Shetal Soni Shetal Soni 1Coralie Di Scala Coralie Di Scala 1,4Tommi Kajander Tommi Kajander 2Claudio Rivera Claudio Rivera 1,4Anastasia Ludwig Anastasia Ludwig 1*
  • 1 Neuroscience Center, HiLIFE, Univeristy of Helsinki, Helsinki, Finland
  • 2 Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland
  • 3 Neuroscience Center, HiLIFE, University of Helsinki, Helsinki, Finland, Helsinki, Uusimaa, Finland
  • 4 Aix Marseille University, INSERM, INMED, 13273, Marseille, France

The final, formatted version of the article will be published soon.

    The neuron-specific K-Cl cotransporter KCC2 maintains low intracellular chloride levels, which are crucial for fast GABAergic and glycinergic neurotransmission. KCC2 also plays a pivotal role in the development of excitatory glutamatergic neurotransmission by promoting dendritic spine maturation. The cytoplasmic C-terminal domain (KCC2-CTD) plays a critical regulatory role in the molecular mechanisms controlling the cotransporter activity through dimerization, phosphorylation, and protein interaction. To identify novel CTD regulatory motifs, we used the Mu transposon-based mutagenesis system to generate a library of KCC2 mutants with 5 amino acid insertions randomly distributed within the KCC2-CTD. We determined the insertion positions in 288 mutants by restriction analysis and selected clones with a single insertion site outside known KCC2 regulatory motifs. We analyzed the subcellular distribution of KCC2-CTD mutants in cultured cortical neurons using immunocytochemistry and selected ten mutants with ectopic expression patterns for detailed characterization. A fluorescent Cl--transport assay in HEK293 cells revealed mutants with both reduced and enhanced Cl--extrusion activity, which overall correlated with their glycosylation patterns. Live-cell immunostaining analysis of plasma membrane expression of KCC2-CTD mutants in cultured cortical neurons corroborated the glycosylation data. Furthermore, the somatodendritic chloride gradient in neurons transfected with the KCC2-CTD mutants correlated with their Cl--extrusion activity in HEK293 cells. Analyzing CTD mutations that modulate KCC2 activity enhances our understanding of its function and is essential for developing novel anti-seizure therapies.

    Keywords: KCC2, SLC12A5, GABA, chloride homeostasis, Mu transposon mutagenesis, KCC2-CTD mutations

    Received: 03 Oct 2024; Accepted: 10 Dec 2024.

    Copyright: © 2024 Uvarov, Fudo, Karakus, Golubtsov, Rotondo, Sukhanova, Soni, Di Scala, Kajander, Rivera and Ludwig. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence: Anastasia Ludwig, Neuroscience Center, HiLIFE, Univeristy of Helsinki, Helsinki, Finland

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