Indicating CaMKII in AMPA Receptor Phosphorylation

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
• Selectively blocking CaMKII inhibits LTP
  induction
• Activated CaMKII enhances AMPA response
• 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?
• Theta-burst LTP induction? immunoblot with
  Abp-T286
• Stable increase in P-T286 phosphorylation maximal
  at 5 mins after induction
• Total autophosphorylation with 32P labeling
  increases more slowly

4
LTP Enhances GluR1 Phosphorylation

• Immunoprecipitate GluR1 from 32P labelled
  hippocampal slices
• Copurified with GluR2/3 assembled receptors
• Significant phosphorylation at 15 and 60 mins

5
CaMKII phosphorylation of AMPAR

• Is AMPAR phosphorylation catalyzed by CaMKII?
• 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
• APV has similar effects

6

• Trypsin Digest peptide map of 32P-AMPAR shows
  enhanced phosphorylation of one site after LTP
• Co-Trasfection of GluR1 with CaMKII in HEK-293
  cells gives identical peptide map
• 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
• This has been shown to enhance AMPA receptor
  response to glutamate
• CamKII inhibition attenuates synaptic
  potentiation after 5-10 min

8
A Look Ahead (if you dare)

• Undertake the same experiment under tetanic
  protocol
• Determine Phosphorylation site on AMPAR and
  requirement in LTP
• Perform learning and memory evaluation of
  aCaMKII(Thr286A) mutant mice