Investigating stimulus- and maturation dependent changes in neuronal K+ /Cl− co-transporter 2 phosphorylation and interaction partners

Abstract: The K+ /Cl− co-transporter 2 (KCC2), is majorly expressed in neurons, is responsible for maintaining Cl− homeostasis inside the cell and is crucial for fast hyperpolarizing GABAA receptor mediated synaptic inhibition. Electrogenic Na+ /HCO3 − co-transporter 1 (NBCe1) is ubiquitously expressed and is the major pH regulator in glial cells, whose role in neurons remains elusive. Ankyrin-G (AnkG) is a scaffolding protein that plays a role in anchoring proteins on neuronal membrane. All three of these proteins have been shown to be regulated by TGF-β signalling. A recent study has identified NBCe1 and AnkG as part of KCC2 interactome. In this study, the primary objectives were to identify the putative functional effects of the KCC2/NBCe1 interaction on baseline and stimulus-induced changes in KCC2 phosphorylation and function, to identify the role of TGF-β in activity-dependent changes in KCC2 phosphorylation and to investigate the role of TGF-β2 signalling in regulating KCC2/AnkG interaction. Human embryonic kidney (HEK)-293 cells stably transfected with KCC2wt, acute adult mouse hippocampal slice cultures, primary hippocampal neurons and organotypic hippocampal slice cultures from wild-type and Slc4a4-/- mice, and hindbrain and forebrain tissue from wild-type and Tgf-β2 deficient mice was used. In HEKKCC2wt cells, NBCs inhibition prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. Nbce1 deficiency, but not functional NBCs inhibition, prevented staurosporine-mediated downregulation of KCC2 phosphorylation at S940 (pKCC2 S940) and at T1007 (pKCC2 T1007) in immature neurons, and downregulated baseline pKCC2 S940. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to wild-type, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. However, functional experiments revealed that KCC2 transport activity was unaffected, and baseline intracellular pH levels were similar between wild-type and Slc4a4-/- neurons. Inhibition of the TGF-β signalling pathway in acute adult hippocampal slice cultures prevented 4AP-mediated downregulation of pKCC2 S940 and T007 and co-immunoprecipitation revealed that loss of TGF-β2 signalling prevents KCC2/AnkG interaction in the embryonic hindbrain. Overall, the present study provides first insights of a putative functional KCC2/NBCe1 interaction, implicating modulation of stimulus mediated phosphorylation, a preliminary role of TGF-β signalling in regulating activity-dependent KCC2 phosphorylation, and a novel description of the dependence of KCC2/AnkG interaction on TGF-β2 signalling- all mechanisms with putative pathophysiological relevance

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2024

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2024
• Urheber: Pethe, Abhishek
• Beteiligte Personen und Organisationen: Roussa, Eleni ; Klugbauer, Axel ; Hecht, Andreas ; Albert-Ludwigs-Universität Freiburg. Abteilung für Molekulare Embryologie ; Albert-Ludwigs-Universität Freiburg. Fakultät für Biologie

Weitere Informationen

• DOI: 10.6094/UNIFR/249192
• URN: urn:nbn:de:bsz:25-freidok-2491928
• Rechteinformation: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:49 MEZ