The excitatory postsynaptic membrane is trans-synaptically bridged to the presynaptic membrane through the interactions of N-Cadherins, the Ephrin ligand and its receptor (EphR), and the binding partners/cell adhesion molecules Neurexin and Neuroligins 1 and 3. Together these molecules stabilize the synaptic cleft and mediate neurotransmission. Glutamate is the predominant and canonical excitatory neurotransmitter released from presynaptic vesicles into the synaptic cleft. Its core excitatory postsynaptic receptors are AMPA, NMDA, and metabotropic glutamatergic receptors (mGluRs).
AMPA receptors (AMPARs) are tetrameric ionotropic glutamatergic receptors. The C-terminal sequences of AMPARs contain unique PDZ domains that allow each subunit to interact with distinct scaffolding proteins, anchoring the receptors to cytoskeletal elements. For example, two subunits of the AMPAR tetramer, GluA2 and GluA3, form direct protein interactions with GRIP1, an adaptor protein with 7 PDZ domains, and PICK1 through their PDZ domains. Importantly, due to incompatible PDZ domains, AMPARs can only indirectly interact with PSD-95, an important post synaptic density protein, via their direct binding to a TARP protein known as stargazin. GRIP1 also binds EphR and GRASP, a Ras guanine nucleotide exchange factor. GRASP inhibits AMPAR targeting and incorporation into the membrane, thereby affecting synaptic plasticity. In addition, neuronal pentraxins (NP1, NARP and NPR) can be secreted presynaptically and may be involved in internalization or clustering of AMPARs. Aside from GRASP and neuronal pentraxins, AMPARs are tightly regulated through phosphorylation events. CaMKII, JNK, FYN, PKC and PKG can all phosphorylate AMPARs, further contributing to synaptic plasticity by influencing AMPAR localization (receptor recycling and translocation from vesicles to the synaptic membrane of both AMPARs and TARPs is regulated by phosphorylation events), and ion channel conductance. The continual exchange of AMPARs occurs via lateral diffusion as well as dynamic phosphorylation of AMPAR, which regulates its trafficking in and out of the cell surface. This may represent molecular mechanisms of long-term potentiation (LTP, i.e. the strengthening of the synapse) and long-term depression (LTD, i.e. the weakening of synapses), respectively. PP1 and PP2B are two phosphatases that act on and inactivate kinases in the excitatory postsynaptic density. LTP and LTD constitute experience-dependent plasticity and play a major role in learning and memory function in the brain.
NMDA receptors (NMDARs) also play a major role in synaptic plasticity. Like AMPARs, NMDARs are ionotropic glutamatergic receptors. When glutamate binds NMDARs, it results in the activation and opening of a non-selective voltage-dependent ion channel. AMPAR-mediated depolarization of the postsynaptic neuron dislodges inhibitory cations from the NMDA pore and allows the flow of Na2+ and Ca2+ into the cell and the flow of K+ out of the cell. The influx of Ca2+ and the resultant activation of CaMKII are the first key steps in achieving LTP. Like AMPARs, NMDARs receptor trafficking from vesicles to the postsynaptic membrane are mediated by resident kinases and phosphatases. However, unlike AMPARs, NMDAR subunits can directly bind PSD-95. Along with the phosphorylation events, this interaction with PSD-95 stabilizes surface expression of NMDARs. PSD-95 is an abundant protein within the excitatory postsynaptic density, an electron-dense cytoplasmic structure that is comprised of hundreds of proteins related to signal transduction and structural regulation of the postsynapse. Of the scaffolding proteins, Homer and Shank are the most abundant. They form a mesh-like matrix and recruit another protein, GKAP, to mediate binding to PSD-95. Together, this tetrameric complex is critical for the structural and functional integrity of the postsynaptic density. Another protein, SynGAP, also binds to the PDZ domain of NMDAR-bound PSD-95. SynGAP is a Ras-GTPase activating protein and plays a role in negatively regulating Ras, thereby mediating NMDAR-dependent control of AMPAR potentiation and membrane trafficking.
Along with AMPARs and NMDARs, mGluRs also mediate glutamatergic neurotransmission. mGluRs are G-protein coupled receptors that transduce signals via the interaction of intracellular G proteins after their large extracellular N-terminal domain binds glutamate. This, in turn, initiates a large intracellular signaling cascade. There have been 8 subtypes of mGluRs identified and stratified into three major groups based on sequence homology, G protein partners, and ligand selectivity. mGluRs exist as dimers and their C-terminal tails interact with Homer, an intracellular protein that bridges mGluRs with IP3Rs to modulate Ca2+ dynamics at the synapse.
In the excitatory postsynaptic density, Ca2+ signaling plays a major role partially due to CamKII activation and subsequent downstream effects. CamKII not only phosphorylates key kinases that are important in synaptic plasticity, but it also binds and crosslinks F-actin filaments. This is thought to both anchor CamKII in spines and to stabilize F-actin bundles to augment spine size. This demonstrates a kinase-independent mechanism by which CamKII can impact synaptic plasticity. In addition, CaMKII phosphorylates Neuroligin 1, increasing its surface expression and promoting the creation of new synapses. Aside from transmembrane receptors, Ca2+ influx into the cytoplasm can also be mediated by IP3Rs, resident receptor in the membrane endoplasmic reticulum. IP3R-mediated Ca2+ release further contributes to CamKII activation and regulation of AMPAR function, thereby also contributing to synaptic plasticity.
The principal inhibitory postsynaptic receptors are GABA receptors (GABARs) and glycine receptors (GLYRs). Both GABARs and GLYRs are members of the ligand-gated ion channel superfamily. They both form heteropentamers and contain 4 transmembrane domains, a large extracellular N-terminal domain, and a large intracellular domain between the 3rd and 4th transmembrane domains. The extracellular N-terminal domain is the site of GABA or Glycine neurotransmitter binding.
The inhibitory postsynaptic membrane is trans-synaptically bridged to the presynaptic membrane via the interaction between Neurexin and Neuroligins 2/3/4, different members of a family of transmembrane synaptic cell adhesion molecules (CAMs). Neuroligin 2 and Neuroligins 3/4, bind to distinct proteins intracellularly, further anchoring the postsynaptic density. During development, Neuroligin 2 interacts with another transmembrane CAM, Slitrk3, through its extracellular domains. Slitrk3 further regulates inhibitory synapse development through the interaction with the axonal receptor protein tyrosine phosphatase, PTPδ. Aside from its role in inhibitory synapse development, the intracellular domain of Neuroligin 2 binds Gephyrin, the principal element in anchoring, clustering, and stabilizing of GABARs and GLYRs in the inhibitory postsynaptic membrane.
Gephyrin is a multimeric hexagonal lattice scaffold that undergoes extensive posttranslational modifications that change its clustering, trafficking and binding properties. Gephyrin directly binds GABARs and GLYRs, polymerized tubulin (i.e. microtubules), and a host of other auxiliary proteins. One such protein is the GDP/GTP exchange factor collybistin, which was shown to promote gephyrin clustering via Cdc42-mediated F-actin clustering. In addition, gephyrin also binds profilin and Mena. Studies suggest that this gephyrin/profilin/Mena/actin complex contribute to cytoskeletal organization within the inhibitory postsynaptic density. Importantly, GABAR activity itself induces the palmitoylation of gephyrin by DHHC-12 leading to increased clustering of gephyrin and an increase in inhibitory synaptic transmission. This illustrates a cyclical feedforward loop between gephyrin organization, GABAR function and inhibitory neurotransmission.
Aside from Gephyrin, Neuroligin 2 also binds MDGA1, a cell-surface molecule that regulates the formation of neuroligin-neurexin connections via steric hinderance of the binding sites on neurexins. MDGA1 expression inhibits formation of inhibitory synapses and serves as a checkpoint of synaptogenesis. On the other hand, Neuroligins 3/4 also contribute to signaling and membrane stabilization by binding to the dystrophin complex (i.e., syntrophin, dystrobrevin, and dystrophin). The exact function of this complex in neurons is still unknown, but studies show that it may serve as a cytoskeletal scaffold for signaling proteins within the inhibitory postsynaptic space.
Similar to excitatory postsynaptic receptor trafficking, trafficking of GABARs is essential for modulation and proper function of inhibitory synapses. GABARs are assembled in the ER then trafficked to the Golgi where they are packaged into vesicles destined for the plasma membrane. The intracellular receptor trafficking of GABARs is mediated by GABARAP, a protein which interacts with its intracellular domain and can be enriched in intracellular compartments. Increase in GABARAP expression increases the surface expression of GABARs. GABARs also undergo extensive endocytosis (following PKC-mediated phosphorylation of GABAR), lysosomal degradation, and recycling. In addition, GABARs can localize extrasynaptically and require shuttling and lateral movement within the membrane to reach their synaptic destinations. This is partially mediated by gephyrin but also by Ankyrin G, a giant ankyrin that is tethered to extrasynaptic GABARs. Ankyrin G inhibits GABAR endocytosis via an interaction with GABARAP, increasing the expression of GABARs and promoting the stability of GABAergic synapses. Importantly, GABAR localization and function can also be modulated by Ca2+ influx through NMDA receptors. This is because a Ca2+ sensitive phosphatase, calcineurin, can directly modulate GABAR phosphorylation status upon increase in intracellular Ca2+ levels. This demonstrates cross-talk between excitatory and inhibitory postsynaptic signaling and further reinforces the importance of intracellular Ca2+ levels in modulating excitation and inhibition.