Abstract by Line Brogaard Palmelund

Ca²⁺/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a multifunctional detector of changes in intracellular Ca²⁺ levels in several Ca²⁺-dependent signalling pathways. These include physiological events such as synaptic plasticity and pathological such as ischaemic cell death. CaMKIIα is activated by Ca²⁺/Calmodulin, and the subsequent Thr286 autophosphorylation regulates the kinase activity independently of Ca²⁺/Calmodulin. Recently, γ-hydroxybutyric acid (GHB) ligands were found to selectively target the CaMKIIα hub domain, responsible for the assembly of holoenzymes. In the hub, the ligands mediates stabilisation with an unknown functional outcome. To characterise the mechanism-of-actions, we aimed to determine the binding mode of the GHB ligands including a study on their high selectivity. Additionally, we hypothesised that the neuroprotective properties of the GHB ligands were mediated by regulation of Thr286 phosphorylation. Lastly, we aimed to characterise dysfunctional CaMKIIα hub variants for their potential as models for functional rescue by GHB ligands. By using site-directed mutagenesis and radioligand binding assay, we validated the key molecular determinants constituting the binding pocket. Moreover, we identified the highly flexible pocket loop and the C-terminal tail as distinct regions involved in the binding of the GHB radioligands. By introducing a CaMKIIα-specific tryptophan into the pocket loop, we successfully obtained GHB ligand binding to CaMKIIβ indicating a significance of the residue composition on this region for binding. To study the response of the GHB ligands on Thr286 autophosphorylation, we established two autophosphorylation assays using recombinant and native CaMKIIα. In contrast to previous reports, the assays did not show any effect of the GHB ligands on Thr286 autophosphorylation. However, the simplified expression systems employed in these assays may not provide a suitable cellular environment for this type of investigation, as several of the downstream targets of CaMKIIα are missing. To characterise the CaMKIIα hub domain variants, we employed a subcellular localisation assay using transfected hippocampal neurons and included an analysis of the neuronal migration upon in utero electroporation. Despite not observing any phenotypic effects of the CaMKIIα variants, Ph-HTBA was found decrease the amount of CaMKIIα R457H in the synaptic site and/or increase the cytosolic amount in the dendritic shaft. However, the mechanism behind this potential neuroprotective effect is unknown and will need further functional elucidation.  Overall, the results from this thesis has provided new insights into the binding modes and selectivity of the GHB ligands, which aid the future development of new drug candidates. Further investigations are needed to elucidate the functional response of the GHB ligands, but the R457H mutant may potentially be used for this purpose.