Two classes of receptors, ionotropic and metabotropic, are responsible for mediating excitatory neurotransmission within the CNS. Ionotropic receptors, commonly referred to ligand-gated ion channels, are responsible for mediating
the fast neurotransmission that occurs at the synapse. Specific binding of a ligand or neurotransmitter to the receptor results in a conformational shift allowing for the flow of ions (i.e. Na+, Ca2+, K+, Cl-) through the channel. Three distinct classes of excitatory ionotropic receptors have been established- α- amino-3-hydroxy-S-methylisoxazole-4-propionic acid (AMPA), N-methyl-D- aspartate (NMDA), and kainate (KA). In contrast to ionotropic receptors are metabotropic receptors which activate a second-messenger system, following binding of a ligand or neurotransmitter. Unlike ionotropic receptors which always mediate excitatory neurotransmission, the second-messenger system associated with metabotropic glutamate receptors (mGluRs) allows them to regulate either inhibitory or excitatory neurotransmission. Eight different mGluRs (mGluR1-8) have been identified and classified into three distinct groups (Group 1 – III). Of particular interest is the ability of kainate receptors (KARs) to mediate both ionotropic and metabotropic transmission, however the focus of the proposed experiments will be on the ionotropic action of KARs.
1.10.1KAINATE RECEPTOR ASSEMBLY AND LOCALIZATION
Kainate receptors (KARs) were identified as a separate class of ionotropic glutamate receptors based on their sensitivity to the agonist kainate (kainic acid), a naturally occurring compound found in seaweed. Studies conducted throughout the early 1990's concluded the presence of five distinct kainate receptor subunits, GluK1 – 5 (Hollmann and Heinemann, 1994), which form functional tetramers. Each of the available KAR subunits contains a large extracellular amino-terminal
domain, involved in subunit assembly, followed by the extracellular ligand binding domain. The membrane region is composed of three transmembrane domains (TM1, TM3, TM4) and a re-entrant P-loop (TM2), which does not fully span the membrane. The M4 domain gives way to the intracellular carboxy-terminus of the subunit (Mayer, 2006).
GluK1 – 5 subunits are divided into two families based on their sequence homology, functionality and agonist affinity. GluK1 – 3 subunits (Bettler et al., 1990; Egebjerg et al., 1991; Bettler et al., 1992; Sommer et al., 1992) and GluK4 – 5 subunits (Werner et al., 1991; Herb et al., 1992; Sakimura et al., 1992) are each about 70% identical within their family, but share only a 40% identity with each other. In addition to differences in the sequence homology of the two groups, GluK1 – 3 subunits can form functional homomeric receptors (Egebjerg et al., 1991; Sommer et al., 1992; Schiffer et al., 1997), while GluK4 – 5 subunits must be co-expressed with GluK1 – 3 (Werner et al., 1991; Herb et al., 1992). Binding assays from recombinant KARs have demonstrated that GluK4 – 5 subunits have significantly higher agonist affinity than do the GluK1 – 3 subunits (Monaghan and Cotman, 1982; Monaghan et al., 1989). In addition to the differences between GluK1 – 3 and GluK4 – 5 subunits, GluK1 and GluK2 are also subject to mRNA editing that alters Ca2+ permeability and sensitivity to intracellular polyamines (Sommer et al., 1991; Rosenthal and Seeburg, 2012). Furthermore, two membrane proteins that interact with KAR subunits were identified, Neuropilin and Tolloid-like 1 and Neuropilin and Tolloid-like 2 (Neto1 and Neto2, respectively) (Zhang et al., 2009; Straub et al., 2011b; Tang et al.,
2011). These proteins have been shown to dramatically alter the basic properties of KARs (Copits et al., 2011; Straub et al., 2011a; Fisher and Mott, 2012, 2013).
KARs composed of GluK2/GluK5 subunits are believed to be the most prominent within the brain (Petralia et al., 1994), thus the majority of studies investigating KAR assembly and trafficking have focused on these two subunits. One check-point involved in proper receptor assembly is the occupancy of the ligand-binding site. Recombinant studies have demonstrated that mutations that reduce agonist affinity results in the subunits being retained within the ER and not inserted into the plasma membrane (Mah et al., 2005; Gill et al., 2009; Fisher and Housley, 2013). For homomeric GluK2 receptors, glutamate binding promotes proper folding resulting in a conformational change that is essential for assembly and trafficking of the receptor (Gill et al., 2009). The GluK5 subunit was found to contain several ER retention motifs that prevented surface expression unless this subunit was bound to a GluK1 – 3 subunit (Gallyas et al., 2003; Ren et al., 2003), a process that promotes formation of heteromeric receptors. Furthermore it was recently determined that agonist binding to the GluK5 subunit alone was sufficient to promote the formation of heteromeric GluK2/5 subunits (Fisher and Housley, 2013). Together these studies demonstrate the complexity of receptor assembly and trafficking as well as represent a key role for both the GluK2 and GluK5 subunits.
Early studies have conclusively demonstrated that mRNA for a portion of kainate receptor subunits is expressed throughout the brain and spinal cord (Wisden and Seeburg, 1993; Bahn et al., 1994; Bettler and Mulle, 1995). Similar
results were noted in the hippocampus, in which mRNA for all of the KAR subunits was also observed, however the expression within the hippocampus varies between regions and cell types. Furthermore, the mossy fiber – CA3 synapse was the first hippocampal region shown to utilize kainate receptors (Castillo et al., 1997; Vignes and Collingridge, 1997). In this region the CA3 pyramidal cells, as well as dentate granule cells, show elevated mRNA levels for GluK2, GluK4 and GluK5. GluK3 mRNA appears to be restricted to dentate granule cells, while interneurons predominately express GluK1, in addition to GluK2 – 3 (Bahn et al., 1994; Bureau et al., 1999; Paternain et al., 2000; Darstein et al., 2003).
Variability in subunit localization, differences in the biophysical properties of the subunits and regulation by Neto1 and Neto2 make it difficult to study KARs
in vivo. Furthermore, these differences become more apparent when attempting to use pharmacological agents to activate or block the receptor, as each subunit may behave differently to agonists and antagonists. As a result of this variability the role of KARs in regulating excitatory neurotransmission, specifically within the diseased brain, is not fully understood.