Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide

Johannes Piepgras; Astrid Rohrbeck; Ingo Just; Stefan Bittner; Gudrun Ahnert-Hilger; Markus Höltje

doi:10.3389/fncel.2022.860823

Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide

Front Cell Neurosci. 2022 Jun 15:16:860823. doi: 10.3389/fncel.2022.860823. eCollection 2022.

Authors

Johannes Piepgras¹, Astrid Rohrbeck², Ingo Just², Stefan Bittner¹, Gudrun Ahnert-Hilger³, Markus Höltje⁴

Affiliations

¹ Department of Neurology, Focus Program Translational Neuroscience and Immunotherapy, Rhine-Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.
² Institute of Toxicology, Hannover Medical School, Hanover, Germany.
³ Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, University of Göttingen, Göttingen, Germany.
⁴ Institut für Integrative Neuroanatomie, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.

Abstract

In primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot^156-181 peptide resulted in the upregulation of glutamate uptake by up to 22% compared to untreated cells. A similar enhancement of glutamate transport was also achieved by the classical phorbol-ester-mediated activation of protein kinase C subtypes. Yet comparable, effects elicited by C3 preparations seemed not to rely on PKCα, γ, ε, or ζ activation. Blocking of tyrosine phosphorylation by tyrosine kinase inhibitors prevented the observed effect mediated by C3bot and C3bot 26mer. By using biochemical and molecular biological assays we could rule out that the observed C3bot and C3bot 26mer-mediated effects solely resulted from enhanced transporter expression or translocation to the neuronal surface but was rather mediated by transporter phosphorylation at tyrosine residues that was found to be significantly enhanced following incubation with either full length protein or the 26mer C3 peptide.

Keywords: C3 transferase; EAAT3; glutamate transporter; phosphorylation; uptake.