Issues to consider
Overall, we should focus on the key differences in GluR1 vs GluR2 expression in terms of how they are trafficked and their unique roles in potentiation. We know that GluR1 is Ca2+ permeable and GluR2 isn't, likewise GluR1 is inward rectifying, GluR2 is not. It should be safe to assume these differences are very meaningful in LTP. Aside from those ion channel differences, GluR1 has a longer C-tail producing differences in trafficking, mobility, and intracellular protein association. Other key LTP-related pathways and mechanisms include: NMDAR-associated calcium influx activates CaMKII, which is rapidly translocated to the active synaptic terminal {{#info: Choquet 2010: NMDAR activation promoted the rapid translocation of aCaMKII::GFP to synaptic sites (marked by Homer1C::DsRed), after which AMPARs were completely immobilized during the 1 min posttranslocation recording period}}. Once situated, CaMKII can increase AMPAR activity at synapses {{#info: Choquet 2010: tCaMKII (constitutively active) promotes immobilization of endogenous GluR1 (containing) AMPARs (both synaptic and extrasynaptic), and to a much lesser extent GluA2 (containing) AMPARs, but CaMKII direct phosphorylation of AMPARs unnecessary for synaptic trapping.}}, but direct phosphorylation is not required for AMPAR expression at synapses {{#info: Malinow (2000): Although such phosphorylaton may enhance the function of synaptic receptors, this phosphorylation does not seem to be required for receptor delivery. tCaMKII can deliver mutated GluR1 (S831A - mutated CaMKII p-site) to the synapse, indicating that some protein(s) other than GluR1 must be substrate(s) of CaMKII (we know Stargazin is one of them)}}. CaMKII perhaps influences AMPAR expression via its PDZ binding domain {{#info: Malinow (2000) found that co(over)expression of tCaMKII and mutate GluR1 (GluR1::T887A::GFP) at its PDZ binding domain completely blocked synaptic response amplitude and rectification, and in fact depressed transmission in hippocampal slice neurons}} {{#info:Choquet 2010 found that the GluA1 - SAP97 interaction unnecessary for CaMKII-dependent synaptic trapping}} {{#info:Choquet 2007 found that GluR2 surface expression was reduced by half when its PDZ binding site was deleted, but lateral diffusion of GluR2 was not changed by this PDZ mutation. Suggesting the PDZ-Binding site of GluR2 controls surface expression, not lateral mobility}} resulting surface expression at synaptic slots; at active synapses CaMKII can also complex with NMDARs, resulting in a sustained increase in activity of both proteins. There strong evidence to suggest that CaMKII works through Stargazin {{#info:Choquet 2007 found that tCaMKII caused a robust immobilization of Stargazin, but not a mutated version of Stargazin (S9A) lacking the CaMKII phosphorylation site, and went on to show the critical importance of Stargazin binding to AMPARs in synaptic trapping}} to increase synaptic trapping of AMPARs {{#info:Choquet 2007 used Stargazin and PSD-95 compensetory mutants to show that the synaptic targeting of Stargazin is dependent on the presence of synaptic PSD-95, and this interaction helped immobilize both GluR1 and GluR2 receptors at synapses}}.
So that is the story, but really to what extent do we know that NMDAR-mediated calcium influx plays dominant role in CaMKII activation. GluR1 homomerics allow calcium to enter the cell too. What evidence is there that GluR1 can't activate CaMKII? Not all synapses even have NMDARs.
To follow up on
- Evidence shows the PDZ site is important for surface expression, not trapping at synapses.
- suggests PDZ is not Stargazin interaction site
- None of the AMPAR subunits bind directly PSD-95 - what other TARP proteins are important for SAP interaction
- the interaction between TARP proteins and AMPARs can be disrupted by glutamate
- Other MAGUKs interact with Stargazin such as SAP-102 and PSD-93
- the interaction of Stargazin with SAP102 and then with increasing level of PSD-95/93 is involved in the higher trapping efficiency of AMPAR at mature synapses
- The PKA phosphorylation of Stargazin C terminus prevents Stargazin binding to PSD-95
- there are apparent GluA1 subunit-specific effect of CaMKII. Although CaMKII triggers the immobilization of both GluA1 and GluA2 containing endogenous AMPARs, it immobilizes homomeric GluA1 but not GluA2 homomeric AMPARs.
- the neuronal pentraxin NARP and NP1 are enriched at excitatory synapses and interact directly with all of the four AMPAR subunits inducing AMPARs surface clustering. NARP and NP1 could thus act as AMPARs stabilizing extracellular factors.
- LTP at the dentate gyrus is independent of CaMKII activity. Also, CaMKII activity is not necessary for LTP early in development at the CA1 region. Further studies will be necessary to determine whether other kinases known to be important for LTP induction, such as PKA, PI3-K, PKC, and MAPK, also trigger AMPAR immobilization.
- Our findings thus raise the possibility that during LTP, CaMKII activation triggers both classical LTP and PPD. It is interesting to note that LTP is frequently accompanied by a decrease in paired-pulse facilitation (PPF)
Zac Email
Here are the different labeling techniques that might be applicable with recombinant expression of AMPARs. Roughly ranked from most to least likely to succeed, separated by large vs small AMPAR N-terminal additions. The references in parentheses are for background on the technique.
- Large AMPAR N-terminal addition
- Small AMPAR N-terminal addition
- AMPAR-FLAG, QD-anti-FLAG
- AMPAR-biotin ligase recognition peptide, QD-biotin + biotin ligase (Lu Ting 2013 PLOSONE)
- AMPAR-peptide A, QD-peptide B, which binds peptide A (Zhang Kodadek 2000 NatBiotech)
- AMPAR-unnatural amino acid azide, QD-propargyl (Chaterjee Schultz 2013 PNAS)
Choquet 2010 CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin
- NMDAR activation promotes rapid translocation of aCaMKII::GFP to synapses, causing AMPAR trapping at 1 min (only synapses with CaMKII translocation)
- tCaMKII (active prion) promotes immobilization of endogenous GluR1 (containing) AMPARs (both synaptic and extrasynaptic), and to a much lesser extent GluA2 (containing) AMPARs.
- CaMKII direct phosphorylation of AMPARs unnecessary for synaptic trapping
- GluA1 - SAP97 interaction unnecessary for CaMKII-dependent synaptic trapping
- Stargazin increased tCaMKII-mediated trapping of recombinant GluA1 (homomeric), but tCaMKII had no effect on mobility of recombinant GluA2 (homomeric)
- Stargazin phosphorylation (by tCaMKII) is necessary for GluA1 trapping; blocking phosphorylation caused AMPAR mobility to significantly increase.
- intriguing finding: GluA1 subunit-specific effect of CaMKII, where it immobilizes recombinant GluA1 but not GluA2 homomeric AMPARs.
- findings consistent with specific role of GluA1 in activity-dependent trafficking - but Stargazin can bind all subunits??
- findings raise possibility that during LTP, CaMKII activation triggers both classical LTP and PPD. Interesting that LTP is frequently accompanied by PPD (opposite of PPF: paired-pulse facilitation)
