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. 2011 Sep 26;590(Pt 1):21–31. doi: 10.1113/jphysiol.2011.213868

Defined criteria for auxiliary subunits of glutamate receptors

Dan Yan 1, Susumu Tomita 1
PMCID: PMC3300042  PMID: 21946847

Abstract

Abstract

Pore-forming subunits of ion channels show channel activity in heterologous cells. However, recombinant and native channels often differ in their channel properties. These discrepancies are resolved by the identification of channel auxiliary subunits. In this review article, an auxiliary subunit of ligand-gated ion channels is defined using four criteria: (1) as a Non-pore-forming subunit, (2) direct and stable interaction with a pore-forming subunit, (3) modulation of channel properties and/or trafficking in heterologous cells, (4) necessity in vivo. We focus particularly on three classes of ionotropic glutamate receptors and their transmembrane interactors. Precise identification of auxiliary subunits and reconstruction of native glutamate receptors will open new directions to understanding the brain and its functions.

Susumu Tomita (left) is Associate Professor in the CNNR program and the Department of Cellular and Molecular Physiology at Yale University School of Medicine. The Tomita laboratory aims to reveal molecular mechanisms controlling synaptic transmission and its regulation, and is especially interested in mechanisms to determine numbers and channel properties of native neurotransmitter receptors at synapses and their dynamism upon neuronal activity. She obtained her PhD in 2000 at the University of Tokyo in the field of Alzheimer's disease. As a post-doc, she studied roles of TARP auxiliary subunits in AMPA receptor activity in David Bredt's laboratory at UCSF collaborating with Roger Nicoll's laboratory. Dan Yan (right) has been working in the Tomita laboratory as a Postdoctoral Research Associate since September 2008. Her main research objective is to elucidate mechanisms regulating the activity of glutamate receptors in synaptic transmission. Her approach is to identify components of native glutamate receptors in the brain and to reconstruct excitatory transmission by expressing those components.

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Introduction

Ion channels are proteins that form pores to allow the selective passage of ions through membranes upon stimulation. For example, changes in membrane potential (voltage) and ligand-binding activate voltage-gated and ligand-gated ion channels (ionotropic receptors), respectively. Pore-forming subunits by themselves show channel activity in recombinant systems. However, recombinant and native channels often display different channel properties, and these discrepancies are solved by the identification and co-expression of channel auxiliary subunits (Isom et al. 1994; Adelman, 1995; Gurnett & Campbell, 1996; Trimmer, 1998; Jackson & Nicoll, 2009; Tigaret & Choquet, 2009). Auxiliary subunits of voltage-gated channels, including voltage-gated sodium channels, voltage-gated calcium channels, and voltage-gated potassium channels, were first identified in the early 1990s (Isom et al. 1994; Adelman, 1995; Gurnett & Campbell, 1996; Trimmer, 1998), but an auxiliary subunit of a ligand-gated channel was not identified until the 2000s (Nicoll et al. 2006; Osten & Stern-Bach, 2006; Ziff, 2007; Milstein & Nicoll, 2008; Payne, 2008; Coombs & Cull-Candy, 2009; Diaz, 2010; Kato et al. 2010b; Tomita, 2010; Jackson & Nicoll, 2011; Opazo & Choquet, 2011). Since then, several proteins have been proposed as auxiliary subunits for ligand-gated ion channels. This review defines an auxiliary subunit of ligand-gated ion channels and focuses in particular on ionotropic glutamate receptors.

Glutamate is a major excitatory neurotransmitter in the vertebrate brain, and three classes of ionotropic glutamate receptors were identified in the early 1990s via expression cloning with cRNA-injected oocytes and homologous gene screening (AMPA-, kainate- and NMDA-sensitive receptors) (Fig. 1) (Nakanishi, 1992; Wisden & Seeburg, 1993; Hollmann & Heinemann, 1994). The cloned pore-forming subunits retained glutamate-gated channel activity in recombinant systems, enabling extensive studies regarding the pore-forming subunit. However, discrepancies in channel properties between recombinant and native receptors have been proposed, some of which were reconciled by co-expression of auxiliary subunits of the receptors (Jackson & Nicoll, 2009; Tigaret & Choquet, 2009).

Figure 1. Structure of ionotropic glutamate receptors and transmembrane interactors/auxiliary subunits.

Figure 1

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Ionotropic glutamate receptors (iGluRs) share similar domain organization, which is N-terminal domain (NTD), ligand-binding domain (LBD), transmembrane (TM) domain, and hydrophobic P-loop. Transmembrane interactors with iGluR are shown with their distinct domain organization.

To elucidate the regulation of ionotropic glutamate receptors in the brain, extensive research has been conducted to identify interactors with ionotropic glutamate receptors. Auxiliary subunits, the topic discussed in this review, also interact with glutamate receptors. How are auxiliary subunits distinguished from interactors? In combination with previously proposed criteria (Isom et al. 1994; Adelman, 1995; Gurnett & Campbell, 1996; Trimmer, 1998; Arikkath & Campbell, 2003; Cannon, 2007), this review proposes four criteria to define an interactor as an auxiliary subunit.

(1) Non-pore-forming subunit

An auxiliary subunit should not be a pore-forming subunit, and the auxiliary subunit in and of itself should not show any ion channel activity, as proposed previously (Isom et al. 1994; Adelman, 1995; Gurnett & Campbell, 1996; Trimmer, 1998; Arikkath & Campbell, 2003; Cannon, 2007).

(2) Direct and stable interaction with a pore-forming subunit

An auxiliary subunit should interact in a direct and stable manner with a pore-forming subunit, as proposed previously (Isom et al. 1994; Adelman, 1995; Gurnett & Campbell, 1996; Trimmer, 1998; Arikkath & Campbell, 2003; Cannon, 2007).

To show direct interaction, ideally, there should be a binding experiment with two purified recombinant proteins in vitro, or the structure of a protein complex at atomic resolution should be examined. However, these experiments are difficult, especially when two transmembrane domains are involved in the interaction. Therefore, purification of a channel complex is optimal for identification of its minimum components, for example silver staining of a channel complex purified from the brain.

To detect stable interactions, native polyacrylamide gel electrophoresis (PAGE) can be a useful approach (Schagger et al. 1994). As a second choice, co-purification of two proteins from heterologous cells and native cells can be performed. However, the native gel approach is preferred, because information regarding the stoichiometry of bound and unbound proteins can be obtained.

(3) Modulation of channel properties and/or trafficking in heterologous cells

An auxiliary subunit should modulate channel properties and/or trafficking when co-expressed with ion channels in heterologous cells, as proposed previously (Isom et al. 1994; Adelman, 1995; Gurnett & Campbell, 1996; Trimmer, 1998; Arikkath & Campbell, 2003; Cannon, 2007).

(4) Necessity in vivo

Auxiliary subunits should be necessary for certain aspects of native ion channel function in vivo. Ideally, disruption of auxiliary subunits by gene-targeting techniques alters expression, channel properties, and/or localization of ion channels in vivo. This newly added criterion will distinguish native auxiliary subunits from proteins behaving as auxiliary subunits only in heterologous cells.

Before discussing the main topic, we would like to emphasize that both interactors and auxiliary subunits are important. They play different roles in controlling channel functions in vivo. For example, a chaperone assists folding or assembly of a protein complex, and its interaction with a channel is transient, but not stable. Scaffolding proteins locate a channel complex at synapses, and their interaction occurs at synapses, but is not stable. Therefore, neither chaperones nor scaffolding molecules are auxiliary subunits. For example, AMPA receptor binds to TARP auxiliary subunit, which interacts with PSD-95 scaffolding protein at synapses (Nicoll et al. 2006; Osten & Stern-Bach, 2006; Ziff, 2007; Milstein & Nicoll, 2008; Payne, 2008; Coombs & Cull-Candy, 2009; Diaz, 2010; Kato et al. 2010b; Tomita, 2010; Jackson & Nicoll, 2011; Opazo & Choquet, 2011). In this case, only TARPs could be called auxiliary subunits by the four criteria proposed above. However, both TARPs and PSD-95 play critical roles in regulating AMPA receptor activity at synapses.

Because of limited space, only transmembrane protein interactors, rather than cytosolic interactors, will be discussed in regard to all three classes of ionotropic glutamate receptors.

AMPA receptors

Among the three classes of glutamate receptors, AMPA receptors gate the most rapidly upon binding to glutamate, mediating fast synaptic transmission and strength. Several transmembrane proteins are proposed to be auxiliary subunits or interactors for AMPA receptors and are discussed here.

Transmembrane AMPA receptor regulatory protein (TARP)

TARP was the first protein described as an auxiliary subunit of the AMPA receptor (Vandenberghe et al. 2005). TARP proteins have four transmembrane domains (Fig. 1) and consist of two classes, Type I (γ-2/Stargazin, γ-3, γ-4, γ-8) and Type II (γ-5 and γ-7). Type I TARPs have a typical PDZ binding domain at the C-terminus, whereas Type II TARPs have a non-canonical PDZ binding domain at the C-terminus. AMPA receptors and TARPs are both expressed strongly in the brain. Reviews describing detailed roles of TARPs as auxiliary subunits of AMPA receptors are available (Nicoll et al. 2006; Osten & Stern-Bach, 2006; Ziff, 2007; Milstein & Nicoll, 2008; Payne, 2008; Coombs & Cull-Candy, 2009; Diaz, 2010; Kato et al. 2010b; Tomita, 2010; Jackson & Nicoll, 2011; Opazo & Choquet, 2011). Therefore, several critical experiments are discussed here regarding each of the four criteria that qualify a protein to be an auxiliary subunit.

(1) Non-pore-forming subunit

Glutamate and kainate evoked currents from heterologous cells expressing the TARP–AMPA receptor complex, but not from cells expressing TARP alone (Chen et al. 2003; Tomita et al. 2004, 2005).

(2) Direct and stable interaction with a pore-forming subunit

Blue native-PAGE (BN-PAGE) gels in combination with Western blotting showed stable and abundant TARP–AMPA receptor complexes in the brain (Vandenberghe et al. 2005; Schwenk et al. 2009). The complex was reconstructed in cRNA-injected oocytes using BN-PAGE (Kim et al. 2010). Furthermore, silver staining of the immunopurified TARP complex demonstrated that the AMPA receptor was the major interactor with TARP (Tomita et al. 2004; Sumioka et al. 2010), and purification of the AMPAR complex identified TARP as a major interactor with the AMPA receptor (Fukata et al. 2005; Nakagawa et al. 2005). However, direct interaction using recombinant TARP and recombinant AMPA receptor has yet to be demonstrated, given the experimentally challenging techniques afforded by binding assays with two transmembrane proteins. Therefore, structural analysis of the AMPA receptor–TARP complex is critical for understanding their direct interaction.

Upon prolonged application of glutamate (longer than 10 min), TARPs physically dissociate from AMPA receptors (Tomita et al. 2004). Subsequent study showed that TARPs functionally decouple from AMPA receptors after desensitization, and this functional decoupling is reversible within 1 s (Morimoto-Tomita et al. 2009). These results indicate that TARPs functionally decouple from AMPA receptors without physical dissociation from AMPA receptors within a time course of synaptic transmission in the brain. Since any two interacting proteins may dissociate in special circumstances like excitotoxicity, unstable or regulated interaction in special circumstances should not be considered to violate criterion (2) if the two proteins form a stable protein complex under native conditions. Indeed, BN-PAGE shows that most of AMPA receptors in the cerebellum have at least one TARP (Vandenberghe et al. 2005; Kim et al. 2010).

(3) Modulation of channel properties and/or trafficking in heterologous cells

TARP enhances the surface expression and channel activity of AMPA receptors in heterologous cells (Fig. 2A). Desensitization and deactivation of AMPA receptors are reduced by TARP co-expression analysed with the ultrafast application of glutamate with the Piezo electric device (Priel et al. 2005; Tomita et al. 2005; Cho et al. 2007; Milstein et al. 2007). In addition, TARPs modulate the pharmacology of AMPA receptors (Fig. 2A) (Tomita et al. 2005, 2007; Menuz et al. 2007; Soto et al. 2007). Type I TARPs modulates both trafficking and channel properties of AMPA receptors (Tomita et al. 2005), whereas Type II TARPs modulate only channel properties of AMPA receptors (Kato et al. 2007, 2008). There is a discrepancy over whether Type II TARP/γ-5 reduces surface expression of GluA2 with Q editing or not (Kato et al. 2008; Soto et al. 2009). Further studies will be required to examine isoform specificity of TARP and AMPA receptor modulation.

Figure 2. Modulation of channel properties in heterologous cells.

Figure 2

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A, glutamate (Glu)- and kainate (KA)-evoked currents were measured from Xenopus laevis oocytes injected with GluA1 cRNA alone or with TARP γ-2/stargazin (STG). Kainate elicited a larger current than glutamate, mimicking native AMPA receptors in the brain (Tomita et al. 2005). B, glutamate-evoked currents were observed in oocytes injected with GluK2 with Neto2, but not GluK2 alone. Neto2 did not modulate GluA1-mediated currents (Zhang et al. 2009). Panel A reproduced from Tomita et al. (2005) and panel B from Zhang et al. (2009) with permission of Elsevier.

(4) Necessity in vivo

TARP γ-2 mutant mice, termed stargazer, showed a loss of AMPA receptor-mediated EPSCs (Fig. 3C) (Hashimoto et al. 1999; Chen et al. 2000). TARP γ-8 knockout mice showed a reduction in the total expression of AMPA receptors as well as the expression of extrasynaptic and synaptic AMPA receptors (Fig. 3A) (Rouach et al. 2005; Fukaya et al. 2006). TARP γ-4 knockout mice showed faster decay kinetics of AMPAR EPSCs (Milstein et al. 2007), and quadruple Type I TARP knockout mice with a disruption in TARP γ-2/3/4/8 died at P0 (Menuz et al. 2009). In addition, TARP γ-7 and γ-2 double knockout mice showed a reduction in AMPA receptors in cerebellar Purkinje cells (Yamazaki et al. 2010). These results support the necessity of TARP for AMPA receptor functions in vivo.

Figure 3. Necessity in vivo.

Figure 3

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A, in the hippocampus of TARP γ-8 knockout mice, a severe reduction in protein expression of CNIH-2 and GluA1/2 was observed without changes in GluK2/3, GluN1 and PSD-95 (Kato et al. 2010a). B, protein expression of Neto1 and GluK5 was reduced in the cerebellum of GluK2 knockout mice (Zhang et al. 2009). C, loss of AMPA receptor-mediated EPSCs was observed without changes in NMDA receptor EPSCs, whereas cerebellar mossy fibre–granule cell synapses in stargazer mice were disrupted. Upper trace, Vh=+40 mV; lower trace, Vh=–70 mV. D, decay kinetics of kainate receptor mediated EPSC; it was significantly faster at hippocampal mossy fibre–CA3 pyramidal cell synapses in Neto1 knockout mice. Panel A reproduced from Kato et al. (2010a) with permission of Elsevier, panels B and D from Straub et al. (2011a), and panel C from Sumioka et al. (2010) with permission of Elsevier.

Although the atomic structure of the TARP–AMPA receptor complex has not been solved, multiple levels of evidence indicate that TARP is an auxiliary subunit of the AMPA receptor. However, the stoichiometry of TARP–AMPA receptors in vivo remains unclear and controversial (Shi et al. 2009; Kim et al. 2010). In recombinant systems, the studies of Shi et al. (2009) and Kim et al. (2010) agree that the stoichiometry could vary from one to four TARPs on each AMPA receptor, depending on the expression level of TARP. However, the stoichiometry of TARP binding to native AMPA receptors could be a minimum of one in cerebellar granule cells or two/four in hippocampal cells. A detailed mechanism in regard to a controlling varying or fixed stoichiometry will be required.

Interestingly, the TARP–AMPA receptor complex is evolutionarily conserved. In C. elegans, the TARP homologue STG-1/2 and the glutamate receptor homologue GLR have been identified (Fig. 1). STG-1/2 together with SOL-1, as discussed below, is absolutely required for GLR-1 activity in vivo (Zheng et al. 2004; Walker et al. 2006a; Wang et al. 2008). Furthermore, whereas TARPs modulate both channel properties and trafficking of AMPA receptors, STG-1/2 modulates only channel properties, but not trafficking, of GLR-1 in recombinant systems and in vivo (Walker et al. 2006a; Wang et al. 2008). Although STG-1/2 relate more closely to Type I TARPs based on the phylogenetic tree (Walker et al. 2006a; Wang et al. 2008), the roles of STG-1/2 are more similar to that of Type II TARPs (Kato et al. 2007, 2008). Evolutionarily conserved and distinct roles of TARPs could shed light on differential regulation of the glutamate receptor.

Cornichon-like (CNIH2/3)

Cornichon-like (CNIH2/3) was identified by proteomic analysis of the native AMPA receptor complex (Schwenk et al. 2009). CNIH2/3 has three transmembrane domains with short intracellular N-termini (6 amino acids) and short extracellular C-termini (1 amino acid) (Fig. 1). CNIH2 and CNIH3 show strong mRNA and protein expression in the hippocampus (Kato et al. 2010a) (Allen Brain Atlas, http://www.brain-map.org/).

(1) Non-pore-forming subunit

Glutamate-evoked currents were observed in stargazer cerebellar granule cells transfected with both TARP γ-8 and CNIH-2, but not in cells transfected with CNIH-2 alone (Kato et al. 2010a).

(2) Direct and stable interaction with a pore-forming subunit

BN-PAGE/Western blotting revealed stable and abundant CNIH–AMPA receptor complexes in the brain (Schwenk et al. 2009). The complex was also detected in a recombinant system using BN-PAGE (Gill et al. 2011). Furthermore, co-immunoprecipitation/mass spectrometry of native AMPA receptor complexes identified CNIH2/3 and TARPs as major interactors (Schwenk et al. 2009). As noted above with the TARP–AMPA receptor complex, examining the direct interaction between recombinant CNIH2/3 and AMPA receptor proteins is technically challenging due to their transmembrane structure. Schwenk et al. (2009) proposed that 80% of AMPA receptors in the brain contained only CNIH2/3, but not TARP. However, subsequent studies by other groups proposed a tripartite complex of TARP γ-8/AMPA receptor/CNIH2 in the hippocampus (Kato et al. 2010a; Gill et al. 2011). The existence of the tripartite complex was mainly supported by three experimental examples. First, the channel properties of the native AMPA receptors could be reconstructed only by co-expression of all three proteins. Second, a significant reduction in protein expression of both AMPA receptors and CNIH2 was observed in the hippocampus of the TARP γ-8 knockout mice. Third, the AMPA receptor and CNIH2 were both specifically immunoprecipitated with an anti-TARP antibody (Kato et al. 2010a; Gill et al. 2011; Schober et al. 2011).

(3) Modulation of channel properties and/or trafficking in heterologous cells

CNIH2/3 slows desensitization and deactivation of AMPA receptors and TARP γ-8–AMPA receptor complex in transfected HEK cells with the Piezo electric device (Schwenk et al. 2009; Kato et al. 2010a; Shi et al. 2010). In addition, CNIH2/3 modulates pharmacology of the AMPA receptor alone and the AMPA receptor–TARP complex (Kato et al. 2010a; Shi et al. 2010; Gill et al. 2011; Schober et al. 2011). Furthermore, CNIH2 enhances surface expression of AMPA receptors in transfected HEK cells (Schwenk et al. 2009; Shi et al. 2010).

(4) Necessity in vivo

TARP γ-8 knockout mice showed a reduction in CNIH2 expression by 20% in the hippocampus, suggesting the existence of a tripartite complex of TARP γ-8–AMPA receptor–CNIH2 in the hippocampus (Kato et al. 2010a). However, phenotypes of a CNIH2/3 knockout mouse have not been reported yet (June 2011) to confirm these findings.

In the heterologous system, multiple studies confirmed that CNIH2/3 can modulate channel properties of AMPA receptors (Schwenk et al. 2009; Kato et al. 2010a; Shi et al. 2010; Gill et al. 2011; Schober et al. 2011). However, it remains unclear whether CNIH2/3 can modulate AMPA receptor activity in the brain. Analysis of CNIH2/3 knockout mice is required to elucidate the roles of CNIH2/3 in vivo. The effects of CNIH2/3 on mEPSC were examined by overexpression of CNIH2/3 in cerebellar granule cells (Kato et al. 2010a; Shi et al. 2010). Whereas CNIH2 did not modulate any factors of mEPSC in cerebellar granule cells from wild-type or stargazer heterozygous mice, i.e. AMPA receptor–TARP γ-2 complexes, CNIH2 slowed the decay kinetics of mEPSC in the TARP γ-8 transfected cerebellar granule cells from stargazer mice. This presumably reflects synaptic AMPA receptor/CNIH2 complexes with TARP γ-8. This discrepancy might suggest that CNIH2 modulates AMPA receptor activity in a TARP isoform-specific manner (Schwenk et al. 2009; Kato et al. 2010a; Shi et al. 2010; Gill et al. 2011; Schober et al. 2011). Detailed analysis of TARP isoform specificity must be conducted to clarify this issue. For example, what is the role of CNIH2/3 in regard to AMPA receptors in vivo? One possibility is that CNIH2/3 dictates the stoichiometry of TARP binding to AMPA receptors (Gill et al. 2011). Interaction of CNIH2/3 and TARP with AMPA receptors is competitive (Gill et al. 2011). Whereas the stoichiometry of TARP binding to AMPA receptors varies from one to four depending on the TARP expression level in heterologous cells (Shi et al. 2009; Kim et al. 2010), TARP stoichiometry could be variable in hippocampus (Shi et al. 2009) or fixed and minimal, i.e. one, in cerebellar granule cells (Kim et al. 2010). Notably, CNIH2/3 expression in cerebellar granule cells has not been confirmed yet. CNIH2/3 might contribute to the difference in TARP stoichiometry in different brain regions.

CKAMP44

CKAMP44 was identified by immunopurification of the native GluA1 AMPA receptor complex from forebrains using iTRAQ between wild-type and GluA1 knockout mice (von Engelhardt et al. 2010). CKAMP44 has a single transmembrane domain with a signal peptide sequence and a potential PDZ binding domain (Fig. 1). CKAMP44 is expressed specifically in the brain and strongly in the dentate gyrus in the hippocampus.

(1) Non-pore-forming subunit

CKAMP44 reduced glutamate-evoked AMPA receptor currents with cyclothiazide, but not kainate receptor and NMDA receptor currents (von Engelhardt et al. 2010).

(2) Direct and stable interaction with a pore-forming subunit

Co-immunoprecipitation experiments using an anti-CKAMP44 antibody showed an interaction between AMPA receptors and CKAMP44 in the brain (von Engelhardt et al. 2010). Since CKAMP44 has been isolated recently, BN-PAGE or immunoprecipitation controls for CKAMP44 knockout mouse have not yet been done.

(3) Modulation of channel properties and/or trafficking in heterologous cells

CKAMP44 co-expression reduced glutamate-evoked AMPA receptor currents and EC50 in cRNA-injected oocytes, without changes in the total and surface expression of AMPA receptors (von Engelhardt et al. 2010).

(4) Necessity in vivo

An increase in the paired pulse ratio was observed in hippocampal dentate granule cells from CKAMP44 knockout mice, but not in CA1 cells (von Engelhardt et al. 2010). Notably, overexpression of CKAMP44 in neurons slowed deactivation and recovery from desensitization of patch membranes from neurons.

It is still too early to describe CKAMP44 as an auxiliary subunit of the AMPA receptor. It is important to reveal the interaction between CKAMP44 and the AMPA receptor as stable or transient. Moreover, the mechanism of current reduction and the role of CKAMP44 in vivo must be determined. CKAMP44 was identified only recently, and therefore increasing knowledge is likely to be revealed in the next few years.

SynDIG1

SynDIG1 was identified by micro-array as one of the highly differentially expressed genes during postnatal cerebellar development (Diaz et al. 2002). SynDIG1 has a single transmembrane domain with a hydrophobic region in its extracellular domain (potentially the membrane re-entrant loop) (Fig. 1) (Kalashnikova et al. 2010).

(1) Non-pore-forming subunit

SynDIG1 has been recently identified (Kalashnikova et al. 2010), and an electrophysiological analysis using heterologous systems has not yet been performed.

(2) Direct and stable interaction with a pore-forming subunit

SynDIG1 can co-immunoprecipitate with AMPA receptors in vivo, but not with NMDA receptors (Kalashnikova et al. 2010). Co-immunoprecipitation experiments using HEK cells transfected with SynDIG1 and GluA2 demonstrated that GluA2 co-immunoprecipitates with full-length and N-terminal-deleted SynDIG1, but not with SynDIG1 lacking 33 amino acids at the C-terminus (Kalashnikova et al. 2010). However, the C-terminal 33 amino acids contain a hydrophobic domain, which is ‘sticky’ in the overexpression system. Therefore, co-immunoprecipitation experiments with a control protein, e.g. GluN1 or GluK2, must be tested in the overexpression system.

(3) Modulation of channel properties and/or trafficking in heterologous cells

SynDIG1 modulates distribution of GluA2 in transfected COS cells (Kalashnikova et al. 2010). Electrophysiological analysis using heterologous systems has not yet been tested.

(4) Necessity in vivo

Knockdown of SynDIG1 by shRNA showed changes in GluA1 and GluA2 distribution together with PSD-95, as well as decreased frequency and amplitudes of miniature EPSCs (Kalashnikova et al. 2010).

It is clear that SynDIG1 modulates AMPA receptor activity at synapses. However, it remains unclear whether SynDIG1 modulates AMPA receptors directly or indirectly. Further analysis will be critical to resolve this issue.

SOL-1

SOL-1 was identified by genetic screening as a gene to suppress the constitutively active GLR-1 mutant, Lurcher, in C. elegans (Zheng et al. 2004). SOL-1 is a single transmembrane protein with an extracellular domain that contains four evolutionarily conserved CUB domains (Fig. 1). In the SOL-1 mutant, loss of GLR-1 activity was shown (Zheng et al. 2004). Although GLR-1 shares low homology in amino acid sequence with AMPA receptors, GLR-1 is defined as an AMPA receptor homologue, because GLR-1 channel properties can be modulated by STG-1 and 2, both homologues of TARP (Walker et al. 2006a; b; Wang et al. 2008).

(1) Non-pore-forming subunit

Glutamate elicited currents from cells expressing the SOL-1–GLR-1–STG and GLR-1–STG complexes, indicating that SOL-1 is not necessary for pore formation (Walker et al. 2006a; b; Zheng et al. 2006).

(2) Direct and stable interaction with a pore-forming subunit

SOL-1 can co-immunoprecipitate with GLR-1, but not the NMDAR homologue NMR-1, in transfected HEK cells (Zheng et al. 2004, 2006). Due to technical limitations in C. elegans, the native SOL-1–GLR-1 complex has not been detected biochemically. Similar to other transmembrane proteins, demonstration of in vitro interaction between two purified transmembrane proteins is difficult.

(3) Modulation of channel properties and/or trafficking in heterologous cells

SOL-1 can modulate channel properties of the GLR-1–STG-1 complex in the cRNA-injected oocyte system (Walker et al. 2006a; b; Zheng et al. 2006; Wang et al. 2008). SOL-1 slows the deactivation/desensitization of cultured muscle cells shown by the piezo electric device (Walker et al. 2006a; b; Wang et al. 2008).

(4) Necessity in vivo

In the SOL-1 mutant, loss of GLR-1 activity was shown (Zheng et al. 2004).

SOL-1 clearly modulates GLR-1 and GLR-1–STG complexes. Strong evidence using genetic approaches and reconstruction in oocytes suggests that SOL-1 is a GLR-1 auxiliary subunit. Interestingly, SOL-1–GLR-1–STG forms a tripartite complex in C. elegans, but a mammalian SOL-1 homologue has not yet been identified. The question remains as to the existence of mammalian SOL-1 homologues. Further genomic and functional analyses will be required to address this question.

Kainate receptors

Kainate receptors are proposed to modulate synaptic transmission at post-synapses and synaptic plasticity at pre-synapses. Several cytosolic interactors are reported to act as interactors with kainate receptors, and their possible roles have been summarized in recent reviews. However, only one family of transmembrane proteins, Neto1 and Neto2, has been proposed to bind to kainate receptors.

Neto1/2

Immunopurification of the kainate receptor complex from rat cerebella and subsequent silver staining/mass spectrometry identified a single protein, neuropilin and tolloid like-2 (Neto2) (Zhang et al. 2009). Neto consists of two isoforms, Neto1 and 2 (Stohr et al. 2002). Neto1/2 is a single transmembrane protein with two CUB domains and one LDL domain in its extracellular region (Fig. 1). Because both neuropilin and tolloid have CUB domains, Netos are termed neuropilin and tolloid-like (Neto) (Stohr et al. 2002). Neto1 and –2 are neuron-specific genes and show complementary expression patterns (Michishita et al. 2004; Straub et al. 2011a).

(1) Non-pore-forming subunit

Neto2 enhances glutamate-evoked currents from GluK2 receptors, but not GluA1 receptors, in cRNA-injected Xenopus oocytes and transfected HEK cells (Fig. 2B) (Zhang et al. 2009; Copits et al. 2011; Straub et al. 2011a). The fact that glutamate failed to evoke currents from heterologous cells expressing Neto2 alone indicates that Neto2 does not form a pore by itself.

(2) Direct and stable interaction with a pore-forming subunit

Proteomic analysis of kainate receptor complexes in the brain identified only Neto2 as a kainate receptor interactor (Zhang et al. 2009). However, as noted above with other transmembrane proteins, an in vitro interaction with the kainate receptor using purified recombinant proteins has not been demonstrated. Furthermore, stoichiometry of Neto2-bound and –unbound kainate receptors remains unclear.

(3) Modulation of channel properties and/or trafficking in heterologous cells

Both Neto1 and Neto2 modulate channel properties, but not surface expression, of GluK2 containing kainate receptors in cRNA-injected oocytes and transfected HEK cells (Fig. 2B) (Zhang et al. 2009; Copits et al. 2011; Straub et al. 2011a; b). On the other hand, kainate receptors modulate surface expression of Neto2 in cRNA-injected oocytes (Zhang et al. 2009). There might be kainate receptor isoform specificity in modulation of kainate receptor trafficking, since Neto2 was shown to modulate surface expression of GluK1 kainate receptors (Copits et al. 2011).

(4) Necessity in vivo

In Neto1 knockout mice, decay kinetics of kainate receptor-mediated EPSCs were substantially hastened, and [3H]kainate binding was reduced in the hippocampal stratum lucidum, where mossy fibre and CA3 pyramidal cells form synapses (Fig. 3D) (Straub et al. 2011a). In GluK2 knockout mice, the total expression of Neto1 and Neto2 was reduced in the hippocampus and cerebellum (Fig. 3B) (Zhang et al. 2009; Straub et al. 2011a). The surface expression of Neto1 and Neto2 was also reduced in the hippocampus and cerebellum of GluK2 knockout mice (Zhang et al. 2009; Straub et al. 2011a).

Similar to TARP interactions with AMPA receptors, the molecular details of the interaction between Neto and kainate receptors remain unclear. Structure–function analysis of these molecules and ultimately atomic structure are required to reveal the mechanism of the interaction.

NMDA receptors

NMDA receptors induce synaptic plasticity due to their calcium permeability via calcium-dependent kinases and phosphatases. Neto1 is proposed to be an auxiliary subunit of the NMDA receptor (Ng et al. 2009), although other reports have described Neto1 and Neto2 as auxiliary subunits of kainate receptors (Zhang et al. 2009; Copits et al. 2011; Straub et al. 2011a; b) (see above).

Neto1

Neto1 was originally identified from a retina-specific gene. Subsequently, ubiquitously expressing splicing isoform and its homologue, Neto2, were identified (Stohr et al. 2002). As noted above, Neto1 is a single transmembrane protein with two CUB domains and one LDL domain in its extracellular domain (Fig. 1). Neto1 is specifically expressed in the nervous system. In the hippocampus, Neto1 is strongly expressed in CA3 pyramidal cells (Michishita et al. 2004; Ng et al. 2009; Straub et al. 2011a), whereas NMDA receptors ubiquitously express in all neurons.

(1) Non-pore-forming subunit

Glutamate failed to bind to Neto1 alone in cRNA-injected oocytes and transfected HEK cells (Zhang et al. 2009; Straub et al. 2011a).

(2) Direct and stable interaction with a pore-forming subunit

Neto1 co-immunoprecipitated weakly with the NMDA receptor subunits NR1, NR2A and NR2B in the brain (Ng et al. 2009). NR2A co-immunoprecipitated with full-length Neto1 and the first CUB domain of Neto1 in transfected HEK cells, indicating that the first CUB domain is sufficient for the interaction between Neto1 and NR2A (Ng et al. 2009).

(3) Modulation of channel properties and/or trafficking in heterologous cells

Modulation of channel properties and/or trafficking has not been tested in heterologous cells.

(4) Necessity in vivo

At the hippocampal SC (Schaffer Collateral)–CA1 synapses, changes in the I–V relationship of the NMDA receptor component and long-term potentiation (LTP) was observed in Neto1 knockout mice (Ng et al. 2009). In addition, reduction of the NR2A subunit in the postsynaptic density (PSD) fraction was observed in Neto1 knockout mice (Ng et al. 2009). Furthermore, second acquisition in the Morris water maze was impaired (Ng et al. 2009).

There is a discrepancy in the role of Neto1 as an auxiliary subunit of glutamate receptors. Whereas Neto2 is proposed to be an auxiliary subunit of kainate receptors (Zhang et al. 2009), Neto1 is proposed to be an auxiliary subunit of both kainate receptors and NMDA receptors (Ng et al. 2009; Zhang et al. 2009). Whereas Ng et al. (2009) showed alteration in channel properties of NMDA receptors in hippocampal CA1 cells in their Neto1 knockout mice, Straub et al. (2011a) showed no changes in NMDA receptor activity in the hippocampal CA3 cells in other Neto1 knockout mice. In addition, Ng et al. (2009) showed co-immunoprecipitation of Neto1 and NMDA receptors, but others failed to detect Neto1/2 interaction with NMDA receptors in the brain (Zhang et al. 2009; Straub et al. 2011a). Both of these studies employed Neto1 knockout mice, but there were critical differences in targeting strategy and the genetic background of the mice. Ng et al. replaced one exon with β-galactosidase gene using 129X1/SvJ ES cells and Straub et al. replaced all exons encoding Neto1 ORF with β-galactosidase gene using C57BL/6NTAC ES cells. Differences in genetic background and gene targeting strategy might have contributed to the conflicting results. Importantly, Neto1 is not detectable in both Neto1 knockout mice (Ng et al. 2009; Straub et al. 2011a).

Cytoplasmic interactors

We here discussed only transmembrane interactors because of limited space. However, a cytoplasmic interactor could be an auxiliary subunit, and indeed β auxiliary subunits of voltage-gated calcium channels and of voltage-gated potassium channels are cytosolic (Isom et al. 1994; Adelman, 1995; Gurnett & Campbell, 1996; Trimmer, 1998). As examples, PSD-95 or KRIP6 may in future be regarded as an auxiliary subunit of kainate receptors (Garcia et al. 1998; Bowie et al. 2003; Laezza et al. 2007, 2008). Both PSD-95 and KRIP6 are not pore forming subunits (criterion (1)), and can bind directly to the cytoplasmic domain of GluK2 kainate receptors (a part of criterion (2)). Furthermore, both PSD-95 and KRIP6 modulate channel properties in heterologous cells co-transfected with GluK2 (criterion (3)). However, stable interaction had not been shown using BN-PAGE or co-purification of protein complex from brain (a part of criterion (2)). Most importantly, necessity in vivo (criterion (4)) has not been shown either. Therefore, at this stage it remains unclear whether PSD-95 or KRIP6 is an auxiliary subunit of kainate receptors or not. More studies, especially using gene-targeting mice and biochemical purification, will be required.

Concluding remarks

Glutamate receptors are major excitatory neurotransmitter receptors and a drug target for neurological disorders. To understand the mechanisms of brain function, reconstruction of the native glutamate receptor in heterologous systems is required. Recent studies have identified the auxiliary subunit of glutamate receptors, which is required for functions of glutamate receptor in vivo. Many interactors for the glutamate receptor have been previously identified; however, it is difficult to distinguish auxiliary subunits from interactors. Here, auxiliary subunits of glutamate receptors are defined by four criteria: non-pore subunit, direct and stable binding, functional receptor modulation in a recombinant system, and necessity in vivo. Differences between auxiliary subunits and interactors can be identified by these four criteria. Precise identification and reconstruction of native glutamate receptors will open new directions to understanding the brain and its functions.

Acknowledgments

The authors thank members of the Tomita lab for helpful discussions. S.T. is supported by NIH MH077939, NS068966, and MH085080.

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