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Title: Mechanisms of αCaMKII synaptic interactions in long-term potentiation and ischaemia
Author: Gabriel, Jonathan Paul
ISNI:       0000 0004 2730 2690
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2012
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Interactions of neuronal αCaMKII in hippocampal CA] pyramidal neurones during ischaemia and long term-potentiation (L TP) are not fully understood. Substrate, CaM and ATP dependencies of αCaMKII were performed using CaM mutants with EF-hands deficient in Ca2+ binding to investigate the interactions of calmodulin (CaM) with aCaMKII during L TP induction. Substrate dependencies demonstrated partial activation by C-terminal EF-hands and demonstrated EF- hand] acts as a V-type activity switch, suggesting how αCaMKII decodes LTP induction dependent intracellular [Ca2] changes. CaM dependencies demonstrated αCaMKII activation regulation by EF- hand 34. ATP dependencies provided evidence for coupling of ATP binding/phosphorylation to C- terminal EF-hand binding, indicating K-type regulation of ATP binding and phosphorylation by CaM at low intracellular [Ca2+]. The mechanism of αCaMKII self-association during ischaemia was studied using EGFP-αCaMKII mutants. ATP binding mutants H282R and K42R, CaM trapping mutant R297E and hydrophobic pocket mutant F293K did not show significant differences in self-association rate from wild type. Hydrophobic pocket mutants F98K and L290K next to the auto-inhibitory helix did not self-associate, indicating importance of this area in cluster formation. Immunogold analysis of wild-type αCaMKII in control, cLTP and ischaemia demonstrated αCaMKII is preferentially post-synaptically localised even in control conditions. Furthermore, results demonstrate aCaMKII translocation to the pre-synaptic area during cL TP. Phospho- Thrz86 αCaMKII immunogold labelling demonstrated that L TP establishment causes transient translocation/autophosphory lation of post-synaptic αCaMKII and also that Thrz86 autophosphorylation is not required for pre-synaptic function. Post-synaptic phospho-Thr-s, αCaMKII labelling demonstrated that this form of αCaMKII is mostly observed during ischaemia. Numbers of aCaMKII clusters observed during ischaemia and chemical L TP were also investigated. Wild-type labelled clusters were significantly increased in ischaemic conditions. There was no significant difference in numbers of phospho- Thrz86 aCaMKII in cLTP and ischaemia, and less phospho- Thr305 αCaMKII clusters were seen in control conditions than in cL TP and ischaemia, indicating' mechanisms of cluster formation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available