1. Indicating CaMKII in AMPA Receptor Phosphorylation A Review of Regulatory Phosphorylation of AMPA-Type Glutamate Receptors by CaM-KII During Long-Term Potentiation Barria, Muller, Derkach, Griffith, & Soderling By group 10

2. Introduction Ca++ influx through NMDA binds CaM which activates CaMKII Ca++ influx through NMDA binds CaM which activates CaMKII Selectively blocking CaMKII inhibits LTP induction Selectively blocking CaMKII inhibits LTP induction Activated CaMKII enhances AMPA response Activated CaMKII enhances AMPA response CaMKII has autophosphorylation site at T286 that can induce Ca++ independent kinase activity CaMKII has autophosphorylation site at T286 that can induce Ca++ independent kinase activity

3. CamKII Autophosphorylation Is CaMKII autophosphorylated during LTP induction and does it remain autophosphorylated throughout LTP? Is CaMKII autophosphorylated during LTP induction and does it remain autophosphorylated throughout LTP? Theta-burst LTP induction  immunoblot with Ab p-T286 Theta-burst LTP induction  immunoblot with Ab p-T286 Stable increase in P-T286 phosphorylation maximal at 5 mins after induction Stable increase in P-T286 phosphorylation maximal at 5 mins after induction Total autophosphorylation with 32 P labeling increases more slowly Total autophosphorylation with 32 P labeling increases more slowly

4. LTP Enhances GluR1 Phosphorylation Immunoprecipitate GluR1 from 32 P labelled hippocampal slices Immunoprecipitate GluR1 from 32 P labelled hippocampal slices Copurified with GluR2/3 – assembled receptors Copurified with GluR2/3 – assembled receptors Significant phosphorylation at 15 and 60 mins Significant phosphorylation at 15 and 60 mins

5. CaMKII phosphorylation of AMPAR Is AMPAR phosphorylation catalyzed by CaMKII? Is AMPAR phosphorylation catalyzed by CaMKII? Treat hippocampal slices with KN-62 and subjected to theta-burst stimulation Treat hippocampal slices with KN-62 and subjected to theta-burst stimulation No effect on STP but attenuated LTP and blocked enhanced phosphorylation of AMPAR and CaMKII No effect on STP but attenuated LTP and blocked enhanced phosphorylation of AMPAR and CaMKII APV has similar effects APV has similar effects

6. Trypsin Digest peptide map of 32 P-AMPAR shows enhanced phosphorylation of one site after LTP Trypsin Digest peptide map of 32 P-AMPAR shows enhanced phosphorylation of one site after LTP Co-Trasfection of GluR1 with CaMKII in HEK-293 cells gives identical peptide map Co-Trasfection of GluR1 with CaMKII in HEK-293 cells gives identical peptide map CaMKII enhances GluR1 responsiveness to exogenous glutamate in HEK cells CaMKII enhances GluR1 responsiveness to exogenous glutamate in HEK cells

7. Conclusions The experiment shows that CamKII autophosphorylation is both necessary and sufficient for GlurR1 phosphoryltaion at Thr286 The experiment shows that CamKII autophosphorylation is both necessary and sufficient for GlurR1 phosphoryltaion at Thr286 This has been shown to enhance AMPA receptor response to glutamate This has been shown to enhance AMPA receptor response to glutamate CamKII inhibition attenuates synaptic potentiation after 5-10 min CamKII inhibition attenuates synaptic potentiation after 5-10 min

8. A Look Ahead (if you dare) Undertake the same experiment under tetanic protocol Undertake the same experiment under tetanic protocol Determine Phosphorylation site on AMPAR and requirement in LTP Determine Phosphorylation site on AMPAR and requirement in LTP Perform learning and memory evaluation of αCaMKII(Thr286A) mutant mice Perform learning and memory evaluation of αCaMKII(Thr286A) mutant mice