Beschreibung:
<jats:p>The K<jats:sup>+</jats:sup>/Cl<jats:sup>‐</jats:sup> co‐transporter 2 (KCC2), a protein exclusively expressed in neurons, facilitates the co‐transport of a K<jats:sup>+</jats:sup> and a Cl<jats:sup>‐</jats:sup> ion across the cell membrane. The developmental shift of GABA<jats:sub>A</jats:sub> receptor mediated responses from depolarizing to hyperpolarizing is mainly due to a developmental upregulation of KCC2. Moreover, in mature neurons, neuronal activity leads to a decrease in surface expression and phosphorylation of Ser940 residue of KCC2, thereby reducing its activity. KCC2 is additionally phosphorylated at Thr906 and Thr1007, which reduces KCC2 surface expression and thus activity.</jats:p><jats:p>Given the important role of neuronal activity in regulating KCC2 expression, we sought to investigate the effect of pilocarpine or 4‐aminopyridine (4AP) treatment on KCC2 expression across different maturation stages of neurons using quantitative RT‐PCR, immunocytochemistry (ICC) and immunoblotting. We also investigated the three phosphorylation sites – Ser940, Thr906 and Thr1007 – of KCC2. Immature neurons (day <jats:italic>in vitro</jats:italic> 4) from embryonic day 18.5 mouse hippocampus were treated with pilocarpine or 4AP (100µM, 60 minutes). The results show that 4AP or pilocarpine treatment has no effect on KCC2 transcript or protein expression, compared to controls. Similarly, ICC and immunoblotting results showed that there was no change in the phosphorylation status of Ser940. ICC results also showed that the phosphorylation status of Thr906 and Thr1007 remained unaltered in immature neurons after pilocarpine treatment.</jats:p><jats:p>In day <jats:italic>in vitro</jats:italic> 17 organotypic hippocampal slices cultured from postnatal day 2 mouse pups, immunoblotting showed that treatment with 4AP did not affect KCC2 protein expression. However, in day <jats:italic>in vitro</jats:italic> 35 organotypic hippocampal slice cultures, 4AP treatment reduced KCC2 whole protein expression.</jats:p><jats:p>These results demonstrate that neuronal activity has a distinctive effect on KCC2 protein as the neurons mature, suggesting a differential regulation of KCC2 protein at different maturation stages.</jats:p>