The potassium-chloride cotransporter KCC2 is a key regulator of chloride homeostasis in neurons of the central nervous system (CNS) and it is critical for the development of fast hyperpolarizing synaptic inhibition. Two full-length isoforms of KCC2, KCC2a and KCC2b, differing by their amino termini have been described. Both isoforms have similar expression levels in neonatal mice, but KCC2b is strongly upregulated in cortical areas during postnatal development, resulting in a developmental shift of GABAergic responses. In contrast to the well-studied KCC2b isoform, the importance of the KCC2a isoform has not yet been demonstrated. My thesis work focuses on characterizing the transcriptional regulation, expression and function of the KCC2 isoforms and a central aim is to elucidate the isoform-specific differences. The molecular mechanisms underlying the regulation of KCC2 gene expression are not yet well understood. Both isoforms show a largely neuron-specific expression pattern and their expression is tightly regulated in development. The KCC2b isoform is also known to be regulated by both normal and pathological neuronal activity. One aim of the present thesis work was to explore the functionality of a conserved E-box site in the KCC2b promoter. Results suggest that the E-box site functions as a binding site for the upstream stimulating factors 1 and 2 (USF1, USF2), two basic helix-loop-helix transcription factors with potentially important roles in brain. Binding of USF proteins to the E-box motif contributes to the upregulation of KCC2b gene expression in immature cortical neurons. Another aim of this study was to compare the postnatal expression patterns of KCC2a and KCC2b proteins in various regions of mouse CNS using immunohistochemistry and isoform-specific antibodies. The cellular expression patterns of KCC2a and KCC2b were largely similar in developing and neonatal mouse. In mature brain, KCC2a is detected in the basal forebrain, hypothalamus, and many areas of the brainstem and spinal cord, but its expression is very low in cortical regions. At the subcellular level, immunoreactivities of the isoforms are only partially colocalized, and KCC2a immunoreactivity, in contrast to KCC2b, is not clearly detected at the neuronal soma surface in most brain areas. Biotinylation experiments suggest that the N-terminal KCC2a epitope might be masked. Results of this thesis work also indicate that the KCC2a isoform, similar to KCC2b, can function as a chloride transporter and decrease the intracellular chloride concentration in cultured neurons. The unique N-terminus of KCC2a includes a SPAK kinase binding site, and the importance of this site and the WNK-SPAK signaling pathway is also explored. Our results indicate that the SPAK kinase is able to bind the KCC2a isoform and to regulate the transport activity of KCC2a more than that of KCC2b.
|Tila||Julkaistu - 2018|
|OKM-julkaisutyyppi||G5 Tohtorinväitöskirja (artikkeli)|
LisätietojaM1 - 76 s. + liitteet
- 3112 Neurotieteet
- 3124 Neurologia ja psykiatria
- 3111 Biolääketieteet