1.4.1 Glutamate receptor subtypes
The amino acid L-glutamate represents the major excitatory neurotransmitter of the central nervous system.44 With the arrival of an action potential (transmitted by voltage-gated sodium and potassium channels) at the presynaptic site of a glutamatergic synapse, voltage-gated calcium channels are opened and calcium flows into the cell.
Upon this stimulus glutamate-filled vesicles are released via exocytosis. After diffusion across the synaptic cleft, glutamate is interacting with several glutamate receptors, which can be assigned to two main groups: metabotropic (G protein-coupled) and ionotropic (ligand-gated ion channel) receptors.116 Metabotropic glutamate receptors mediate the slow excitatory neurotransmission and are involved in multiple biochemical pathways.117,118 The fast excitatory neurotransmission (on a millisecond time scale)119 is mediated by ionotropic glutamate receptors, which are ligand-gated cation channels.120 Ionotropic glutamate receptors are further subdivided into NMDA receptors and the two non-NMDA receptors AMPA receptor and kainate receptor. Their names originate from pharmacologic agonists that selectively bind to and activate the corresponding receptor:
N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainic acid (KA), which are all structurally related to the endogenous agonist glutamate (see Figure 5).
Figure 5: Chemical structures of L-glutamate and its analogues NMDA, AMPA and kainate.
1.4.2 AMPA receptors
Molecular cloning of glutamate receptors has contributed greatly to the understanding of the structure and function of AMPA receptors (AMPARs).47,121,122 Further information was obtained by several crystal structure studies of AMPAR subtypes, which have been published during the last 10 years.123–126 Four subunits have been identified (GluR1-GluR4, also named GluA1-GluA4 after AMPA), which are encoded
N+ O
O -O -O
kainate H H
by the genes Gria1-4. The subunits
share a sequence homology of approximately 70%.
composed of an extracellular amino
domain (D1 and D2), the transmembrane domain (consisting of three membrane spanning domains M1, M3 and M4
and a cytoplasmic carboxy-terminal domain
Figure 6: Schematic model of the domain structure of a single receptor subunit from Rogawski).127 The subunit is composed of an extracellular amino
and D2) domain that represents the binding site for the endogenous agonist glutamate domain (comprising three membrane
a cytoplasmic carboxy-terminal domain.
sites for noncompetitive antagonists
The long extracellular amino
positions,128 is suggested to be involved in subunit dimerization and subtype assembly.129 Also on the extracellular site is the ligand binding site
represents the binding site for the endogenous agonist glutamate as well as for AMPA.
It has been suggested that the agonist initially interacts with the D1 lobe, whereupon the D2 lobe moves towards the D1 lobe and interacts with the ligand
change is transmitted via the linker sequences to the transmembrane domain causing the channel to open.130,131
S2-M4 the binding sites for noncompetitive antagonist
The subunits, which posses a length of about 900 amino share a sequence homology of approximately 70%.47 All of the four
acellular amino-terminal domain (N-terminus), a ligand
2), the transmembrane domain (consisting of three membrane and M4, and one intramembraneous re-entrant loop M2) terminal domain (C-terminus), as depicted in Figure
Schematic model of the domain structure of a single receptor subunit of AMPARs ( s composed of an extracellular amino-terminal and ligand
that represents the binding site for the endogenous agonist glutamate, a transmembrane comprising three membrane-spanning domains M1, M3 and M4, and a re-entrant
terminal domain. In the region of the linker sequences S1-M1 and S2 are assumed.
amino-terminal domain, which is N-glycosylated is suggested to be involved in subunit dimerization and subtype
on the extracellular site is the ligand binding site (D1 and D2)
represents the binding site for the endogenous agonist glutamate as well as for AMPA.
that the agonist initially interacts with the D1 lobe, whereupon the D2 lobe moves towards the D1 lobe and interacts with the ligand. This conformational change is transmitted via the linker sequences to the transmembrane domain
130,131
Within the region of the linker sequences S1 for noncompetitive antagonists are suggested. It is
a length of about 900 amino acids, subunits are , a ligand-binding 2), the transmembrane domain (consisting of three
membrane-entrant loop M2), Figure 6.
of AMPARs (modified terminal and ligand-binding (D1 , a transmembrane entrant loop M2), and M1 and S2-M4 binding
glycosylated at several is suggested to be involved in subunit dimerization and subtype-specific and D2), which represents the binding site for the endogenous agonist glutamate as well as for AMPA.
that the agonist initially interacts with the D1 lobe, whereupon the . This conformational change is transmitted via the linker sequences to the transmembrane domain, thus linker sequences S1-M1 and It is assumed that
those negative allosteric modulators stabilize the conformation of the receptor by hindering the linker domains to transfer the conformational change onto the transmembrane domain and thus impair the opening of the channel.132 The transmembrane domain comprises three hydrophobic domains that are spanning the membrane (M1, M3 and M4) and a fourth domain (M2), which represents an intramembraneous re-entrant loop. This re-entrant loop forms the ion channel pore.133 Within the intracellular carboxy-terminal domain the four subunits exhibit the largest sequence differences. This region interacts with many different proteins, and thus, among other functions, is responsible for targeting the receptor to synapses.134
All of the four AMPAR subunits exist in two variants, called flip and flop, which are products of alternative splicing.121 This flip/flop region is located on the extracellular site in close proximity to the transmembrane domain indicated as M1 in Figure 6. It is encoded by neighbored exons of the subunit gene, which comprise 115 bp. Among different subunits these segments are quite similar, exhibiting differences in the peptide sequence between flip and flop in 9 to 11 amino acids. The flip and flop variants are present in different expression levels during the development of the brain and also exhibit a distinct, but partly overlapping expression pattern throughout diverse brain structures.135 They functionally differ from each other by their kinetic properties: in general, the flop variant desensitizes faster than the flip variant in the presence of glutamate.121,136,137
A functional AMPAR that exhibits two agonist binding sites is composed of four subunits forming a tetrameric receptor structure, which consists of two dimers of the subunits GluR1 to GluR4.47,138,139 While homotetramers represent functional receptors, native receptors are almost exclusively heterotetramers (consisting of two different subunits each in dimer pairs).140 The assembly of AMPARs varies depending on developmental stage and subcellular localization. However, the majority of AMPARs in the adult brain appears to consist of GluR1/GluR2 and GluR2/GluR3 subunit combinations.141,142
1.4.3 The AMPA receptor subunit GluR2
Among the four subunits, the GluR2 subunit plays a central role for AMPARs. It is widely expressed in the central nervous system, being present within the majority of all
AMPARs.47,135,142–144
GluR2 is the only subunit that carries a so-called Q/R-editing site, which is located in the re-entrant loop forming the ion channel pore (indicated as M2 in Figure 6).145–147 This site has an essential function in the regulation of cation permeability of the channel. By posttranscriptional RNA-editing the genetically encoded amino acid glutamine (Q) at position 607 is exchanged by the amino acid arginine (R) in almost all GluR2 subunits. This is mediated by the enzyme adenosine deaminase ADAR2, which is converting adenosine to inosine by hydrolytic deamination, thereby changing the codon CAG to CIG.148 This inosine is read by RNA-dependent RNA-polymerases as guanosine, which changes the codon to CGG.
Subsequently, arginine (CGG) instead of glutamine (CAG) is integrated into the channel forming domain. Due to the positive charge and the steric hindrance by this residue, AMPARs possessing the edited GluR2 subunits are impermeable for calcium and hence only allow monovalent ions (sodium and potassium) to pass the channel.146,149,150
In genetically modified mouse models lacking the GluR2 subunit it could be shown that this subunit has an integral role in development and function of the brain. In the absence of GluR2, the mice show several behavioral abnormalities and an overall increased mortality.151,152 Furthermore, it was demonstrated that mice, which express the unedited GluR2 subunit (heterozygous), exhibit a particular phenotype: due to unhindered calcium permeability they develop epileptic seizures and die shortly after birth.153,154 Similar observations were made with mice (homozygous) lacking the editing-responsible enzyme ADAR2.148
AMPARs represent the major mediator of excitatory neurotransmission and thus are integrally involved in the generation and spread of epileptic seizures.127 Given the fact that the GluR2 subunit is present in the majority of AMPARs together with its dominant role in calcium permeability and the effects observed in mice lacking edited GluR2 subunits, it becomes evident that this subunit crucially contributes to the physiological functioning of AMPARs and thus very likely also in their role in seizure propagation.
Consequently, it might be conceivable that a potential interaction of LEV and its analogues with AMPARs in a negative allosteric manner could contribute to their antiepileptic effects. Due to a much lower abundance than the highly expressed SV2A protein the detection of these receptors might be much more difficult and hence require more sensitive techniques than those applied in previous studies.