In their 2010 study Choquet coexpressed tCaMKII with HA:GluA1 lacking the PDZ-binding domain (HA-GluA1D7) and found that HA-GluA1D7 mobility at synapses was strongly reduced by tCaMKII - their measure was diffusion rate and synaptic trapping, which to me is a weaker measure than Malinow's methods which directly test the functional electrophysiological properties conferred to the synapse by GluR1
results show LTP and CaMKII activity drive GluR1 to synapses by mechanism requiring GluR1 and PDZ proteins
Does tCaMKII-GFP enhanced transmission?
To examine effect of elevated CaMKII activity, we used tCaMKII-GFP.
Genes delivered to neurons in organotypically cultured hippocampal slices, using Sindbis virus
* Neurons expressing trans-genes identified by GFP and whole-cell recordings.
expression of construct increased tCaMKII activity in BHK cells.
In neurons expressing construct GFP was detected in dendritic arbors and spines.
we measured synaptic responses in two nearby neurons, one with tCaMKII-GFP one WT.
tCaMKII-GFP enhanced synaptic transmission but with no effect on rectiﬁcation
Does tCaMKII enhance transmission via GluR1?
We used ephys assay to examine if increase in AMPAR-mediated transmission was due to delivery of receptors to synapses.
The current-voltage (I-V) relationship of AMPARs is determined by GluR2 subunit (GluR2 has linear I-V relations; GluR1 homomerics are rectified at 140 mV). Most AMPARs in hippocampal pyramidal cells contain the GluR2 subunit.
We overexpressed GluR1::GFP subunit in hippocampal slice neurons. Most resulting recombinant AMPARs lacked GluR2. Recombinants were functional and showed complete inward rectification in HEK293 cells. Thus, incorporation of these recombinant receptors into synapses would be expected to increase rectification of synaptic responses.
Can CaMKII activity drive recombinant GluR1-GFP into synapses?
GluR1-GFP is widely distributed throughout dendritic arbors, but little is incorporated into synapses in the absence of activity.
In agreement with this, expression of GluR1-GFP had no effect on amplitude or rectification.
To determine if CaMKII activity could drive recombinant GluR1-GFP into synapses,
we coexpressed GluR1-GFP and tCaMKII using an internal ribosomal entry site (IRES) construct.
BHK cells expressing this construct showed increased constitutive tCaMKII activity, and slices expressing this construct showed GluR1-GFP expression.
Pairwise recordings comparing infected and noninfected cells showed infected cells had enhanced transmission, due to increase of tCaMKII activity.
Notably, infected cell transmission showed increased rectification, indicating a contribution of the homomeric GluR1-GFP to transmission.
This effect on rectification was due to coexpression of the two proteins, because transmission onto cells expressing either tCaMKII or GluR1-GFP alone had rectification comparable to that in uninfected cells.
These results show that tCaMKII activity induces the insertion of homomeric GluR1-GFP into the synapse.
Is GluR1 phosphorylation by CaMKII sufficient to drive LTP?
GluR1 is phosphorylated by CaMKII at Ser831 during LTP.
To examine if direct phosphorylation of the receptor at this site is required for delivery, we substituted Ser831 with Ala, thus creating GluR1(S831A)-GFP.
This mutation did not block delivery.
Expression of this construct alone changed neither amplitude nor rectification, and coexpression with tCaMKII produced potentiated transmission that showed the same increase in rectification as that seen with GluR1-GFP-IREStCaMKII
Is the TGL (PDZ consensus) sequence of GluR1 important for its synaptic trafficking/rectification when tCaMKII is overexpressed
The subcellular localization of many membrane proteins is controlled by associations with a class of proteins containing PDZ domains.
In particular, the cytosolic C-terminus of such surface proteins has a PDZ sequence that when mutated prevents associations. GluR1 has this C-terminus TGL consensus sequence.
We mutated the GluR1 sequence from TGL to AGL, creating GluR1(T887A)-GFP
GluR1(T887A)-GFP expressed in HEK293 cells formed functional AMPARs with normal rectification.
GluR1(T887A)-GFP expressed in hippocampal neurons was detected in dendrites, and showed no effect on transmission when expressed alone.
Coexpression of GluR1(T887A)-GFP and tCaMKII in hippocampal slice neurons completely blocked synaptic response amplitude and rectification, and in fact depressed transmission
Does evoked LTP do the same thing as tCaMKII overexpression, in mediating GluR1 synaptic trafficking/rectification?
To determine if LTP delivers AMPARs to synapses through a similar mechanism, we examined LTP in cells expressing GluR1-GFP.
Whole-cell recordings were obtained from cells expressing or not expressing GluR1-GFP.
LTP was induced with a pairing protocol.
We added APV to the bath 30 min after potentiated transmission was measured, in order to isolate pure AMPAR–mediated responses.
The holding membrane potential was then switched to measure rectification of the AMPAR–mediated responses.
Similar to the effect of coexpressed CaMKII and GluR1-GFP, rectification was increased after LTP in cells expressing GluR1-GFP compared to cells not expressing GluR1-GFP.
Is the PDZ GluR1 binding domain important during LTP?
We next examined the effects of GluR1(T887A) on LTP.
As shown above, GluR1(T887A) has a mutated PDZ-interaction site that completely blocks the potentiation produced by tCaMKII.
we recorded synaptic responses from cells expressing either GluR1-GFP or GluR1(T887A)-GFP.
After LTP pairing, GluR1-GFP control cells displayed stable potentiation lasting at least 50 min.
GluR1(T887A)-GFP cells displayed a very different response after LTP pairing: these cells had short-lasting potentiation that decayed over 20 min and after 45 min the responses were significantly depressed from baseline.
In 4 of the 21 experiments with GluR1(T887A), a control (nonpaired) pathway was monitored, which did not show depression
Here, we generated electrophysiologically tagged receptors to monitor their synaptic delivery during LTP and increased tCaMKII. In the absence of plasticity-inducing stimuli, we saw no evidence for their contribution to transmission (consistent with previous results indicating that in the absence of evoked activity, GluR1 is retained within the dendrite).
Upon coexpression with constitutively active tCaMKII or following LTP induction, we see that tagged receptors contribute to transmission, indicating their delivery to synapses.
Previous studies indicate that LTP induction increases the CaMKII-dependent phosphorylation of GluR1 at Ser831. Although such phosphorylaton may enhance the function of synaptic receptors, this phosphorylation does not seem to be required for receptor delivery: tCaMKII can deliver GluR1(S831A)-GFP to the synapse. Our results indicate that some protein(s) other than GluR1 must be substrate(s) of CaMKII (Choquet: Stargazin) and participate in the regulated synaptic delivery of AMPARs.
The most surprising of our results relate to the effects of GluR1(T887A). This protein forms functional receptors and has no detectable effects on basal synaptic transmission. However, this mutant receptor can block the effects of tCaMKII and LTP (24). This has several implications:
It reinforces the view that CaMKII and LTP act through similar mechanisms.
It indicates that both CaMKII-potentiation and LTP exert their effects through GluR1
It indicates that an interaction between GluR1 and a protein with a PDZ domain plays a key intermediate in these forms of plasticity
GluR1(T887A) depresses transmission, but only after increased CaMKII or LTP.
This last finding suggests that activity enables the mutant protein to interrupt a constitutive delivery of endogenous AMPA-Rs (25).
These results demonstrate that incorporation of GluR1-containing AMPA-Rs into synapses is a major mechanism underlying the plasticity produced by activation of CaMKII and LTP. This process requires phosphorylation of protein(s) other than GluR1. Furthermore, this delivery requires interactions between the COOH-terminus of GluR1 and PDZ domain proteins.