Regulation of NMDA receptors by serine proteases tissue plasminogen activator (tPA) and plasminogen plasmin

Table of Contents Acknowledgements ii 1.1 Glutamate Receptors in the Mammalian Central Nervous System 2 1.3 NMDA Receptors in the Brain: Localization and Architecture 3 1.3.1 The NMDA r

Title Page

REGULATION OF NMDA RECEPTORS BY

SERINE PROTEASES TISSUE PLASMINOGEN ACTIVATOR (tPA) AND

PLASMINOGEN/PLASMIN

NG KAY SIONG

B Appl Sci (Hons.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY

NATIONAL UNIVERSITY OF SINGAPORE

2011

Acknowledgements

First and foremost, I would like to express my greatest gratitude to my supervisor, Dr Low Chian Ming for giving me invaluable advice and guidance in the PhD project throughout the course of this postgraduate degree I am deeply indebted

to his time and patience and also the constant encouragement he has given me

I would also like to thank my co-supervisor, Prof Peter Wong Tsun Hon, who has been providing assistance and advice whenever required

Thanks also go out to my fellow lab members, Ms Cheong Yoke Ping and Ms Zhang Yi Bin for their technical support; Ms Karen Wee Siaw Ling and Ms Leung How Wing for the constant intellectual discussion and encouragement I am also very appreciative of past lab members Dr Ng Fui Mee, Dr Rema Vazhappilly, Dr Vivien Chow and Ms Lim Peiqi, who shared their technical expertise and not forgetting Ms Chen Jing Ting and Ms Noella Anthony for their technical assistance

I would also like to express my appreciation to our collaborators, Prof Stephen F Traynelis (Emory University, Atlanta, GA) and Dr Hiroyasu Furukawa (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) for all the valuable advice

My sincere thanks also go to my thesis examiners for spending their precious time on my thesis

Special thanks also go out to my two mentors, Assoc Prof Tok Eng Soon and

Dr Alvin Teo for their support and assistance

Lastly, I would like to thank my family for their support and encouragement The completion of this thesis would not have been possible without the support of many people I sincerely thank all who have helped in making this postgraduate course a success

Table of Contents

Acknowledgements ii

1.1 Glutamate Receptors in the Mammalian Central Nervous System 2

1.3 NMDA Receptors in the Brain: Localization and Architecture 3

1.3.1 The NMDA receptor gene families and their localization 4

1.3.2.4 Carboxyl terminal domain (CTD) 11

1.4 NMDA Receptor Channel Properties and Pharmacology 13

1.5.2 NMDA receptors residing at the postsynaptic membrane 21

1.7.2 tPA/plasminogen system in the brain 33

1.8.1 Structural modulation: Spine modelling 36

1.8.2 Molecular modulation: Synaptic plasticity 36

1.8.3 Molecular modulation: The NMDA receptor 37

1.8.3.1 tPA promotes neurotoxicity through the NMDA

receptors 37 1.8.3.2 Plasmin cleavage of NMDA receptors 38

1.8.3.3 LRP and the NMDA receptor 39 1.8.3.4 tPA and NR2B-containing NMDA receptors 40

CHAPTER 2 tPA-Induced Cleavage of the NR2B Subunits 43

tPA cleaves rat brain lysate NR2B subunit 51

Antibody epitope mapping 52 tPA cleaves the recombinant fusion protein MBP-ATD2B 58

CHAPTER 3 Plasmin Cleavage of NR1 and NR2B Subunits 69

Plasmin degrades NR2B 72 Plasmin degrades NR1 73 tPA cleavage of NR2B is independent of plasmin 77

CHAPTER 4 Functional Consequence of Truncated NR2B-Containing

ATD-truncated NR2B forms functional NR1/NR2B- ∆ATD-R67 receptors

with reduced ifenprodil sensitivity 87

Truncated NR2B reduces glycine potency 89

Truncation of NR2B-ATD changes D-cycloserine efficacy and potency 89

CHAPTER 5 tPA-Induced Decrease of Synaptic NR2B Subunits 98

tPA decreases NR2B protein levels in the synaptic fraction 105

tPA does not alter NR1 and NR2A protein levels 108

Reference 130

List of Publications

1) Wee XK, Ng KS, Leung HW, Cheong YP, Kong KH, Ng FM, Soh W, Lam Y, Low CM (2010) Mapping the high-affinity binding domain of 5-substituted benzimidazoles to the proximal N-terminus of the GluN2B subunit of the NMDA receptor Br J Pharmacol 159:449-461

2) Ng KS, Leung HW, Traynelis SF, Wong PTH, Furukawa H, Low CM Ectodomain cleavage on the NR2B subunit by tissue plasminogen activator results in a functional truncated NMDA receptor with reduced ifenprodil and glycine affinities (In preparation)

Abstracts

1) Ng KS, Wong PTH, Low CM (2008) ‘Yin and Yang’ of FDA-approved busting recombinant tissue plasminogen activator (tPA): Its proteolytic cleavage

clot-of NR2B subunit clot-of NMDA receptor

(2nd Taiwan/Hong Kong(CU)/Singapore Meeting of Pharmacologists, Kaohsiung, Taiwan, Nov 2008 (Poster presentation))

2) Low CM, Wee XK, Ng KA, Leung HW, Cheong YP, Kong KH, Ng FM, Soh

WQ, Y Lam (2008) Benzimidazole derivatives bind at sub-nanomolar concentrations to recombinant protein of the NR2B amino-terminal domain of NMDA receptor Soc Neurosci Abstr 131.4/D8

(38th Annual Meeting of Society for Neuroscience, Washington DC, USA 2008 (Poster presentation))

3) Ng KS, Wong PTH, Low CM (2007) NR2B subunit of NMDA receptor is a new substrate for tissue plasminogen activator Soc Neurosci Abstr 678.17/F29

(37th Annual Meeting of Society for Neuroscience, San Diego, USA 2007 (Poster presentation))

4) Ng KS, Traynelis SF, Wong PTH, Low CM (2007) Anti-Clotting Agent, Tissue Plasminogen Activator (tPA), cleaves NR2B Subunit of NMDA Receptor in Mammalian Brain

(Office of Life Sciences Conference, National University of Singapore, Singapore 2007 (Poster presentation))

Summary

Tissue plasminogen activator (tPA) is an endogenous serine protease that is found in the vascular system and the central nervous system The tPA/plasminogen proteolytic cascade which converts plasminogen to plasmin through tPA cleavage plays a critical role in dissolving blot clots and helps to maintain vascular patency In the central nervous system, the tPA/plasminogen system is also involved in many processes ranging from synaptic plasticity to neurodegeneration In particular,

increasing evidence implicate tPA as an important neuromodulator of the N-methyl-Daspartate (NMDA) receptors The aim of this thesis is to examine the modulation of NR2B-containing NMDA receptors by the tPA/plasminogen system

-Through the analysis of tPA-treated rat brain lysates, I found that tPA can degrade the NR2B subunits of the NMDA receptors and this tPA-induced degradation was independent of plasmin Peptide sequencing studies performed on the cleaved-off products obtained from the tPA treatment of a recombinant fusion protein containing the amino-terminal domain (ATD) of NR2B, revealed that tPA-mediated cleavage occurred at arginine 67 (Arg67) located in the ATD Hence, I sought to examine how the deletion of a short peptide proximal to valine 68 (Val68) in the NR2B subunit, could alter NMDA receptor function

Electrophysiological studies on Xenopus laevis oocytes which heterologously

expressed NR1 with the ATD-truncated form of NR2B (NR2B-ΔATD-R67) revealed

a reduction in ifenprodil sensitivity In addition, the potencies of glycine and Dcycloserine were reduced Furthermore, the efficacy of D-cycloserine was enhanced when the amino acids 28-67 at the proximal end of the NR2B-ATD was deleted Although the underlying mechanisms of the findings are unknown, these findings

-revealed that the amino acids proximal to Val68 could harbor critical determinants that could be important for the allosteric modulation of NMDA receptor channel properties

It is unknown whether putative tPA-induced NR2B-ATD cleavage of the NR2B

or other forms of modulatory mechanisms of tPA on the NMDA receptors can change the NR2B-containing NMDA receptors levels in different subcellular compartments This paradigm was examined through the acute tPA treatment of P14 whole hippocampi and subjecting treated-hippocampi to subcellular fractionation The subsequent analysis of the different subcellular compartments revealed that tPA treatment led to a decrease in synaptic NR2B subunit levels in the hippocampus

In addition to examining the direct modulatory role of tPA on NR2B-containing NMDA receptors, the proteolytic effect of plasmin on NMDA receptors was also investigated Both NR1 and NR2B were found to be proteolytic substrates of plasmin

My results demonstrated that the ATD, S2 and carboxyl-terminal domain (CTD) of NR1 may harbor potential plasmin cleavage sites, which are mostly consistent with the putative cleavage sites reported by other laboratories In addition, I found that the NR2B subunit can be cleaved by plasmin at two potential sites residing in the CTD New insights into the modulation of NR2B-containing NMDA receptors by the tPA/plasminogen system were presented in this thesis Further studies into the underlying mechanisms engaged by tPA in the modulation of NMDA receptors would enable us to have a better understanding of the multi-faceted roles of tPA in the brain (500 words)

List of tables

Table 1.1 Competitive NMDA receptor antagonists and their binding affinity to

Table 1.2 Selected roles of proteases in the brain 29

Table 2.1 Alignment of critical residues around the scissile peptide bonds of

Table 2.2 Concentrations of tPA used in various research reports 66

Table 5.1 NMDA receptor subunits protein levels after tPA (20 g/ml)

treatment 112

List of figures

Figure 1.1 Classification of glutamate receptors 2

Figure 1.2 The different isoforms of the NR1 subunit 5

Figure 1.3 The tetrameric NMDA receptor structure and the topology of a

Figure 1.5 Phosphorylation sites residing in the CTD of NMDA receptor

subunits 28 Figure 1.6 Differential surface regulation of NR2B-containing NMDA

Figure 1.7 Thrombin-mediated mechanism for potentiation of synaptic NMDA

Figure 1.8 Fibrinolysis involving tPA and plasminogen 33

Figure 1.9 The LRP-PSD-95-NMDA receptor complex 41

Figure 2.3 Antibody epitope mapping suggests ATD cleavage 56

Figure 2.4 The recombinant fusion protein MBP-ATD2B 57

Figure 2.5 tPA cleaves the MBP-ATD2B fusion protein 60

Figure 2.6 The strand representation of the crystal structure of apo-NR2B ATD

Figure 3.3 α2-antiplasmin does not prevent tPA-induced degradation of NR2B

proteins 78 Figure 4.1 Ifenprodil inhibition of NR2BWT and NR2B-∆ATD-R67 containing

Figure 4.2 Electrophysiological characteristic of heterologous NR1/NR2BWT

and NR1/NR2B-∆ATD-R67 receptors in Xenopus oocytes 90

Figure 4.3 NR2B- ATD-R67 changes DCS potency and efficacy 91

Figure 4.4 The strand representation of the crystal structure of apo-NR2B ATD

and the truncated ATD (3JPW.pdb) (Karakas et al., 2009) 94

Figure 5.1 Schematic of the subcellular fractionation procedure for hippocampi

Figure 5.2 Acute tPA treatment on P14 hippocampi results in a decrease in

Figure 5.3 Protein levels of the various NMDA receptor subunits in the

synaptic (PSD) fraction after tPA (20 g/ml) treatment 109

Figure 5.4 NR1 protein levels in the different subcellular fractions 110

Figure 6.1 Proposed modulation of NMDA receptors by the tPA/plasminogen

system 122

Abbreviations

a.a Amino acids

AMPA -amino-3-hydroxy-5-methyhl-4-isoazolepropionic acid

AP2 Adaptor protein-2

ATD Amino-terminal domain

CaMKII Ca2+/Calmodulin dependent protein kinase II

Cdk5 Cyclin-dependent kinase 5

cDNA Complementary deoxyribonucleic acid

CKII Casein kinase II

CNS Central nervous system

CTD Carboxyl terminal domain

DCS D-cycloserine

ECM Extracellular matrix

EPSP Excitatory postsynaptic potential

ER Endoplasmic reticulum

ERK Extracellular-regulated kinase

FDP Fibrin-degraded products

GABA γ-aminobutyric acid

GluRs Glutamate receptors

iGluRs Ionotropic glutamate receptors

LB Luria-Bertani

LBD Ligand-binding domain

LIVBP Leucine/isoleucine/valine binding protein

LRET Luminescence resonance energy transfer

LRP Low-density lipoprotein receptor-related protein

LTP Long-term potentiation

MAGUK Membrane-associated guanylate kinase

MALDI-TOF Matrix-assisted laser desorption/ionization – time of flight

MAPK Mitogen-activated protein kinase

mGluRs Metabotropic glutamate receptors

MMP Matrix metalloproteinase

mRNA messenger ribonucleic acid

MS Mass spectrometry

nACh Nicotinic acetylcholine

NMDA N-methyl-D-aspartate

PAI-1 Plasminogen activator inhibitor-1

PAR Protease-activated receptor

PKA Protein kinase A

PKC Protein kinase C

PMSF phenylmethylsulfonyl fluoride

PN-1 Protease nexin-1

PSD Postsynaptic density

PTK Protein tyrosine kinase

RNA Ribonucleic acid

SAP Synapse-associated protein

SEM Standard error of the mean

TEVC Two-electrode voltage-clamp

tPA Tissue-type plasminogen activator

uPA Urokinase-type plasminogen activator

UTR Untranslated region

CHAPTER 1 Introduction

CHAPTER 1

Introduction

1.1 Glutamate Receptors in the Mammalian Central Nervous System

Neural transmission is a critical component of the many processes regulating the normal functioning of the mammalian central nervous system (CNS) Facilitating neural transmission, neurotransmitters play a vital role in CNS development, gene expression, synapse formation and cell division/migration (Cavelier et al., 2005) At the majority of excitatory synapses in the CNS, the amino acid L-glutamate is a key modulatory neurotransmitter (Niswender and Conn, 2010) L-glutamate modulates neuronal and synaptic transmission by targeting, binding and activating glutamate receptors (GluRs) found abundantly at synapses GluRs consist of two families, namely the metabotropic glutamate receptors (mGluRs), which modulate synaptic transmission through G-protein signaling (Kew and Kemp, 2005; Pinheiro and Mulle, 2008); and the ionotropic glutamate receptors (iGluRs), which mediates the vast majority of rapid excitatory synaptic transmission in the CNS (Figure 1.1) (Dingledine et al., 1999; Kew and Kemp, 2005; Chen and Wyllie, 2006)

Figure 1.1 Classification of glutamate receptors

Glutamate Receptors

GluR2 GluR3 GluR4

GluR5 GluR6 GluR7 KA1 KA2

NR2A NR2B NR2C NR2D

NR3A NR3B

Delta

1

2

1.2 Ionotropic Glutamate Receptors

The iGluRs are ligand-gated ion channels that are pharmacologically classified

into three types, namely N-methyl-D-aspartate (NMDA) receptors, hydroxy-5-methyhl-4-isoazolepropionic acid (AMPA) receptors and 2-carboxy-3-carboxymethyl-4-isopenylpyrodidine (kainate) receptors (Dingledine et al., 1999; Kew and Kemp, 2005; Lodge, 2009) (Figure 1.1) Additionally, the delta receptors, often termed the orphan receptors, are also classified under the iGluR family based on sequence homology (Dingledine et al., 1999; Yuzaki, 2003; Schmid and Hollmann, 2008) Each class of iGluRs consists of various subunits which are further classified based on gene sequence homology (Dingledine et al., 1999)

-amino-3-1.3 NMDA Receptors in the Brain: Localization and Architecture

Named after the original agonists used to activate them selectively, NMDA receptors are a class of ligand-gated ion channels that are expressed in many parts of the brain in both neonatal and adult brains Activation of NMDA receptors allow influx of calcium ions (Ca2+) into neurons which will trigger various downstream events and signalling cascades (Dingledine et al., 1999) Excitatory in nature, the NMDA receptors play essential roles in neuronal development, synaptic transmission and synaptic plasticity (Ulbrich and Isacoff, 2008) However, over-activation of NMDA receptors can lead to excessive Ca2+ influx and neuronal death due to excitotoxicity (Dirnagl et al., 1999) In addition to excitotoxicity, NMDA receptors have been implicated in many pathophysiological conditions and neurological diseases such as ischemic stroke, pain, epilepsy, Alzheimer’s disease and

schizophrenia (Villmann and Becker, 2007) Hence, there is a huge interest in the research of NMDA receptors as potential therapeutic targets

1.3.1 The NMDA receptor gene families and their localization

The NMDA receptor family consists of seven subunits, namely NR1, NR2A, NR2B, NR2C, NR2D, NR3A and NR3B These subunits are classified under three subfamilies based on gene sequence homology (Figure 1.1) and are differentially expressed both temporally and spatially in the brain (Dingledine et al., 1999)

1.3.1.1 The NR1 subunit

The NR1 subunit is the only member in the NR1 subfamily Encoded by a single gene consisting of twenty-two exons, the NR1 subunit is expressed ubiquitously in the brain Molecular diversity of the NR1 subunit is achieved through alternative RNA splicing of three independent exons in the NR1 gene, namely exon 5

in the N terminus (N1 cassette) and exons 21 (C1 cassette) and 22 (C2 cassette) in the

C terminus (Figure 1.2) (Hollmann et al., 1993; Hollmann and Heinemann, 1994; Zukin and Bennett, 1995) Splicing out the C2 cassette within exon 22 removes the original stop codon and results in a new reading frame that encodes an alternative cassette C2’ before a stop codon is reached Up to eight distinct NR1 isoforms can be obtained through RNA splicing Based on Hollmann’s nomenclature, NR1-1 refers to the clone which contains both C1 and C2 cassettes, NR1-2 lacks the C1 cassette, NR1-3 lacks the C2 cassette and NR1-4 lacks both the C1 and C2 cassette The letter

‘a’ and ‘b’ indicate the absence and presence of exon 5 respectively (Hollmann et al., 1993)

The expression of different splice variants of the NR1 subunit are temporally and spatially regulated (Laurie and Seeburg, 1994a) The expression of most of the NR1 splice isoforms commences at embryonic day 14 and peaks during the third postnatal week before it decreases slightly upon adulthood The localization of the various splice isoforms were observed to be established during birth and remained throughout brain development (Laurie and Seeburg, 1994a) The NR1-a and NR1-2 isoforms are expressed homogenously throughout the brain, while the NR1-b isoforms are localized in the sensorimotor cortex, thalamus and neonatal caudate The NR1-1 isoforms are found in rostral areas such as the cortex, caudate and hippocampus, while the NR1-4 isoforms are localized in the complementary caudal regions such as the thalamus, colliculi and the cerebellum Low levels of the NR1-3 variant can only be detected in postnatal cortex and hippocampus

Figure 1.2 The different isoforms of the NR1 subunit.1 Differential splicing of

exons 5, 21 and 22 can give rise to eight different NR1 subunit isoforms

C2 ’ C1

NR1-4b NR1-4a NR1-3b

NR1-1a

NR1-2a NR1-2b NR1-3a NR1-1b

While no NR2A splice variants has been discovered, the other NR2 subunit genes undergo alternative splicing Five isoforms for the mice NR2B gene have been reported and they can be obtained from the alternative splicing of non-coding exons 1’, 1 and 2, which are found in the 5’ untranslated region (UTR), upstream of the transcriptional start site (Klein et al., 1998; Tabish and Ticku, 2004) At least four NR2C isoforms have been detected in both the rat and human brains (Suchanek et al., 1995; Daggett et al., 1998; Rafiki et al., 2000) Likewise, two NR2D splice variants have been proposed (Wenzel et al., 1996)

In the embryonic brain, expression of NR2A and NR2C are non-existent while expression of NR2B transcript can be found in areas such as the cortex, hippocampus, thalamus and spinal cord, together with NR2D in the midbrain structures (Monyer et al., 1994; Mony et al., 2009a; VanDongen, 2009) At birth, NR2B transcript expression is widespread in the brain, with particularly high expression in the cortex, hippocampus, striatum and thalamic nuclei while NR2D expression is prevalent in the midbrain structures At this stage, low levels of NR2B can also be found in the cerebellum At around postnatal day P7-10, expression of NR2B peaks, together with an increase in NR2A expression and a drop in NR2D expression Increase of NR2A expression occurs in areas such as the cortex,

hippocampus and the cerebellum From P12, NR2B expression begins to decrease in the cerebellum and eventually leads to a complete disappearance in the developed cerebellum Both NR2A and NR2B expression continue to peak in the third postnatal week before they decline to moderate levels in the adult brain NR2C expression markedly increases from P7 to P12 and peaks in the developed cerebellum (granule cells) and remains as the dominant subunit in the cerebellum

1.3.1.3 The NR3 subunits

There are currently two members in the NR3 subfamily and they are the NR3A and NR3B subunits (Ciabarra et al., 1995; Sucher et al., 1995; Nishi et al., 2001) Within the subfamily, NR3B is closely related to NR3A, with approximately 47% similarity in amino acid sequence However, there is a 17-21% homology when compared to the NR1 and NR2 subfamilies (Chatterton et al., 2002) Two variants of the NR3A gene have been reported in the rat brain, however, only one form is found

to be expressed in the human brain (Eriksson et al., 2001)

From birth, NR3A transcripts and proteins can be found in the spinal cord, brainstem, thalamus, amygdala, lateral olfactory tract, hippocampus and the cortex (Ciabarra et al., 1995; Sucher et al., 1995; Wong et al., 2002) The levels of NR3A increase from late embryonic development and remain elevated until the second postnatal week when the levels in all areas, except the lateral olfactory tract, decrease drastically

NR3B mRNA is localized mostly in the mouse spinal cord and brainstem and its expression levels remain fairly constant during development from birth to the adult brain (Nishi et al., 2001; Matsuda et al., 2002) In addition, NR3B mRNA can also be found in the hippocampus and the cerebellum in adult rats (Andersson et al., 2001) NR3B proteins are expressed in the brainstem in adult mouse brains and in the

hippocampus, cortex, striatum, cerebellum and spinal cord of the adult rat (Matsuda et al., 2003; Wee et al., 2008)

1.3.2 Subunit topology

All NMDA receptor subunits have similar membrane topologies, a modular architecture, which is characteristic of all members of the iGluRs (Traynelis et al., 2010) Comprising of distinct functional regions, each subunit consists of an extracellular amino-terminal domain (ATD), a S1S2 ligand-binding domain (LBD), three trans-membrane domains (M1, M3 and M4) with a re-entrant pore loop (M2) and a cytoplasmic carboxyl terminal domain (CTD) (Traynelis et al., 2010) (Figure 1.3)

1.3.2.1 Amino-terminal domain (ATD)

The amino-terminal domain (ATD), found extracellularly, is made up of approximately the first 350 – 400 amino acids (a.a.), excluding the signal peptide (Herin and Aizenman, 2004; Madry et al., 2007a; Yuan et al., 2009a) Through structural homology modeling, it was proposed that the ATD of NMDA receptor subunits might have a clamshell-like structure, similar to that of the bacterial periplasmic leucine/isoleucine/valine binding protein (LIVBP) (Masuko et al., 1999; Paoletti et al., 2000) The clamshell-like structure architecture of the ATD of NMDA receptor subunits was subsequently verified through crystallographic studies on the ATD of NR2B by Karakas and co-workers (Karakas et al., 2009) The elucidation of the ATD architecture thus allows better comprehension of the functional mechanics of the ATD of NMDA receptor subunits, particularly as a regulatory domain

Figure 1.3 The tetrameric NMDA receptor structure and the topology of a single

subunit (Left) The tetrameric NMDA receptor, a membrane receptor, is an assembly

of the obligatory NR1 subunit and NR2 and/or NR3 subunits The activation of NMDA receptors requires the binding of glutamate to NR2 subunits and glycine to NR1 subunit, together with the removal of Mg2+ upon membrane depolarization Activated NMDA receptors allow the influx of Ca2+ (Right) The topology of a

subunit is shown in details A NMDA receptor subunit has several domains, which include the amino-terminal domain (ATD), ligand-binding domain (S1-S2 LBD), trans-membrane domains (M1, M3 and M4), a re-entrant loop (M2) and a carboxyl terminal domain (CTD) Agonists glutamate and glycine bind to the S1-S2 LBD region, while modulators such as ifenprodil and zinc bind to the ATD The CTD interacts with various scaffolding proteins and contains various posttranslational modification sites

Intracellular

NR1 NR2

Zn 2+ (NR2A), Ifenprodil (NR2B)

CTD

Studies of the NMDA receptor function and pharmacology have led to the discovery that a variety of subunit-specific ligands can target the ATD of NMDA receptors and regulate the receptor function allosterically (Paoletti and Neyton, 2007; Mony et al., 2009a) Antagonistic ligands, such as Zn2+ and ifenprodil, have been found to inhibit NMDA receptor channel activity (Westbrook and Mayer, 1987; Legendre and Westbrook, 1991) and their binding sites have been mapped to the ATD

of NR2A and NR2B subunit, respectively (Paoletti et al., 1997; Low et al., 2000; Choi

et al., 2001; Perin-Dureau et al., 2002) (see section 1.6.1) In addition to playing a role

in regulation of the NMDA receptor channel activity, the ATD of NMDA receptors has been proposed to be involved in receptor assembly and receptor trafficking (Perez-Otano et al., 2001; Herin and Aizenman, 2004; Qiu et al., 2009)

1.3.2.2 S1S2 ligand-binding domain (LBD)

The S1S2 ligand-binding domain (LBD) is the other domain found extracellularly This domain is an important part of the NMDA receptor as it is the activation centre for NMDA receptors The binding site for agonist glutamate resides

in the LBD of NR2 subunits, while the LBD of NR1 binds co-agonist glycine (Herin and Aizenman, 2004) The LBD consists of two parts: S1, a region which starts after ATD and ends before M1 and S2, which is the extracellular loop between M3 and M4 (Paoletti et al., 2000) (Figure 1.3) The S1S2 LBD was found to be related to the bacterial lysine/arginine/ornithine binding protein (LAOBP), a clamshell-like structure, through homology modeling and hence predicted to exist as a clamshell-like structure, similar to the ATD (Sutcliffe et al., 1996; Paoletti et al., 2000) In 2003, Furukawa and Gouaux crystallized the S1S2 LBD of NR1 and experimentally verified the bilobed or clamshell structure of the LBD (Furukawa and Gouaux, 2003)

The glutamate and glycine binding sites are also binding sites for competitive antagonists such as 2-amino-5-phosphonopentanoate (AP5) and 7-chlorokynurenic acid (7-CKA) (Paoletti and Neyton, 2007) (see section 1.4.2.2) Recently, it has been reported that the integrity of the glycine binding site acts as an important functional quality checkpoint for the trafficking of assembled NMDA receptors to the cell surface (Kenny et al., 2009)

1.3.2.3 Transmembrane domain

There are four hydrophobic segments, M1 – M4, in each NMDA receptor subunit (Figure 1.3) The proposed architecture of the transmembrane domains were based on the hydrophobicity profile of different neurotransmitter-gated ion channels (Mori and Mishina, 1995), N-glycosylation sites which are markers of extracellular localizations (Roche et al., 1994; Taverna et al., 1994) and the location of the LBD (Kuryatov et al., 1994; Mori and Mishina, 1995) Thus, the three transmembrane segment model with a re-entrant loop, proposed for kainate binding proteins and GluR1 subunit, was deemed appropriate for NMDA receptors (Mori and Mishina, 1995; Dingledine et al., 1999)

The cytoplasmic-facing re-entrant loop M2 and the transmembrane M3 segment represent major pore-lining domains in GluRs, including NMDA receptors (Sobolevsky et al., 2004) In addition, the M3 segment has been suggested to be a transduction intermediate which couple ligand binding to channel opening (Jones et al., 2002; Yuan et al., 2005; Blanke and VanDongen, 2008)

1.3.2.4 Carboxyl terminal domain (CTD)

The carboxyl terminal domain (CTD) is the final modular segment of the NMDA receptor subunit and is found intracellularly The CTD interacts with a wide

range of signaling proteins, enzymes and scaffold proteins (Ryan et al., 2008) One of the most prominent interacting partners of the CTD of NR2 subunits is the membrane-associated guanylate kinase (MAGUK) super-family of proteins which includes members such as the postsynaptic density (PSD) -95 protein and synapse-associated protein (SAP) -102 (Sheng, 2001) In addition to anchoring and localizing NMDA receptors to the cell surface at postsynaptic sites, the PSD-95 proteins also facilitate in coupling the NMDA receptors to cytoplasmic signaling pathways, by bringing cytoplasmic signal-transducing enzymes closer to the receptors (Sheng and Pak, 2000; Sheng, 2001; Hardingham, 2009) In addition, the CTD is important for post-translational modification of the NMDA receptor as it contains critical amino acid residues which are substrates for phosphorylation (Lee, 2006; Chen and Roche, 2007) (See section 1.6.2) Determinants of surface expression of NMDA receptors, such as endoplasmic reticulum (ER) retention signals, are also found in the CTD (Standley et al., 2000; Scott et al., 2001; Zhang et al., 2008; Horak and Wenthold, 2009) Recently, the CTD was reported to be involved in long-term potentiation (LTP), an electrophysiological phenomenon believed to underlie learning and memory in the brain (Foster et al., 2010) Thus, the CTD plays an important role in the normal functioning of NMDA receptors

1.3.3 Structure of the NMDA receptor

It is widely accepted that NMDA receptors are hetero-tetrameric assemblies of two obligatory NR1 subunits together with two NR2 subunits, where one NR2 subunit can be replaced by one NR3 subunit (Laube et al., 1998; Dingledine et al., 1999; Cull-Candy et al., 2001; Furukawa et al., 2005; Köhr, 2006; Mony et al., 2009b; Sobolevsky et al., 2009) (Figure 1.3) It has been demonstrated that the NMDA

receptors could be formed by dimerization of a pair of either homomeric NR dimers

or heteromeric NR dimers (Schorge and Colquhoun, 2003; Papadakis et al., 2004; Schüler et al., 2008; Rambhadran et al., 2010) Although the exact NR dimer composition is unclear, isolated crytallographic studies provided some insights into the dimer formation paradigm Isolated domains of AMPA and kainate receptor subunits, including the full homotetrameric GluR2 AMPA receptor, were found to form homodimers (Armstrong and Gouaux, 2000; Sun et al., 2002; Jin et al., 2009; Kumar et al., 2009; Sobolevsky et al., 2009), while isolated LBD of NMDA receptor subunits were found to be heteromeric (Furukawa et al., 2005) In addition, the crystallographic study on the ATD of NR2B suggests that the NR2B ATD does not form homodimers With the progress in crystallographic studies, it should not take long before the complete heteromeric structure of the NMDA receptor is elucidated

1.4 NMDA Receptor Channel Properties and Pharmacology

Unlike the AMPA and kainate receptors, binding of glutamate alone will not activate the NMDA receptors Activation of NMDA receptors is unique, as it requires the simultaneous binding of the agonist glutamate to the LBD of NR2 subunits and the co-agonist glycine to the LBD of NR1 subunits, in addition to the relief of the voltage-dependent magnesium (Mg2+) block via membrane depolarization (Mayer et al., 1984; Johnson and Ascher, 1987; Kleckner and Dingledine, 1988) The influx of extracellular Ca2+ through the open channel pore can trigger various intracellular signalling cascades, which can lead to altered neuronal function (Dingledine et al., 1999)

The composition of the NMDA receptors critically determines the ion channel

subunits, the non-NR1 subunits confer distinct pharmacological and physical properties to the NMDA receptor, resulting in a diversity of NMDA receptor function (Cull-Candy et al., 2001; Erreger et al., 2007)

1.4.1 Channel properties

1.4.1.1 NR2-containing NMDA receptors

Although glutamate binds to the NR2 subunits during NMDA receptor activation, glutamate has different affinities towards different NR2 subunits and the order of affinity is NR2B > NR2A or NR2D > NR2C (Laurie and Seeburg, 1994b) In addition, the identity of NR2 subunits can affect the glycine affinity to the NR1 subunit (Ikeda et al., 1992; Kutsuwada et al., 1992; Chen et al., 2008; Yang and Svensson, 2008) While glycine exhibited similar affinities for NR2B or NR2D containing receptors, glycine has almost ten-fold higher affinity for NR2C containing receptors NR2A-containing receptors have the lowest sensitivity for glycine (Laurie and Seeburg, 1994b) This suggests that there is allosteric coupling between the NR1 and NR2 subunits In addition to agonists affinity, many other properties of the NMDA receptor such as channel gating kinetics (Vicini et al., 1998; Erreger et al., 2004), channel open probability (Wyllie et al., 1998; Chen et al., 1999; Popescu et al., 2004; Erreger et al., 2005; Dravid et al., 2008; Gielen et al., 2009), tonic Mg2+ block (Monyer et al., 1994; Kuner and Schoepfer, 1996; Qian et al., 2005; Clarke and Johnson, 2006) and desensitization (Erreger et al., 2004; Erreger and Traynelis, 2005) are also affected by the identity of the NR2 subunits

1.4.1.2 NR3-containing NMDA receptors

NMDA receptors containing the NR3 subunit in general are found to have decreased current amplitude, lower Ca2+ permeability, decreased Mg2+ sensitivity and

a reduced unitary conductance (Ciabarra et al., 1995; Sucher et al., 1995; Das et al., 1998; Nishi et al., 2001; Perez-Otano et al., 2001; Chatterton et al., 2002; Matsuda et al., 2002; Matsuda et al., 2003; Tong et al., 2008) In agreement with the properties of NR3-containing NMDA receptors, Nakanishi and colleagues reported NR3A-containing NMDA receptors might be neuroprotective (Nakanishi et al., 2009)

While many groups were unable to obtain functional NR1/NR3-containing receptors in mammalian cells (Nishi et al., 2001; Matsuda et al., 2002), Smothers and co-workers reported robust glycine-activated currents in HEK293 cells and subsequently reported that expression of functional NR1/NR3 receptors in mammalian cells is dependent on the NR1 isoform (Smothers and Woodward, 2007, 2009) Nonetheless, NR1/NR3 functional excitatory glycine receptors expressed in

Xenopus oocytes are activated by glycine alone, insensitive to glutamate and NMDA

receptor antagonists such as Mg2+, MK801 and have low Ca2+ permeability (Chatterton et al., 2002; Madry et al., 2007b)

binding of glutamate to NR2 subunits and glycine to NR1 subunits were first proposed from mutagenesis studies and subsequently verified by crystallographic studies of the NR1 and NR2A LBD (Dingledine et al., 1999; Furukawa and Gouaux, 2003; Furukawa et al., 2005)

The glycine site residing in the LBD of NR1 is in fact not exclusive to glycine binding D-serine, found abundantly in the brain with similar concentration to glycine, is the ‘other’ endogenous co-agonist that binds to the glycine site in NR1 subunits (Hashimoto et al., 1995; Schell et al., 1995; Mothet et al., 2000; Shleper et al., 2005) In fact, D-serine has high affinity for the glycine binding site and has up to threefold higher affinity than glycine (Matsui et al., 1995; Furukawa and Gouaux, 2003) However, despite numerous reports on D-serine-mediated processes, there is

no consensus on the relative importance and contributions of glycine versus D-serine

in mediating NMDA receptor function (Herman Wolosker, 2008)

1.4.2.2 Competitive antagonists

Competitive antagonists bind to the ligand binding sites and deprive the natural agonists of their binding site, thereby preventing the activation of the NMDA receptors Most synthetic glutamate site antagonists are conformationally constrained, -amino carboxylic acids containing an -phosphonic group (Kew and Kemp, 2005) (Table 1.1) D-AP5 is commonly used because it shows strong selectivity for NMDA receptors over the other iGluRs (Paoletti and Neyton, 2007) Like glutamate, these compounds have different affinities for the different NR2 subunits, typically in the order NR2A > NR2B > NR2C > NR2D (Table 1.1) Glutamate site antagonists in general cannot selectively distinguish and inhibit unique NR2 subtypes as the difference in subunit affinity is usually less than tenfold (Feng et al., 2005; Paoletti and Neyton, 2007) (Table 1.1) The Novartis compound NVP-AAM077 was initially

reported to be a NR2A-specific antagonist with more than hundredfold selectivity for NR2A-containing receptors compared to NR2B-containing receptors (Auberson et al., 2002) However, other groups have found that the NR2A selectivity by NVP-AAM077 has been overestimated and hence, it is likely that NVP-AAM077 may not

be able to clearly discriminate between NR2A and NR2B containing receptors (Frizelle et al., 2006; Neyton and Paoletti, 2006)

Glycine site competitive antagonists bind to the LBD of NR1 subunits Likewise, many glycine site antagonists have been identified and a fair number of them, such as 7-chlorokynurenic acid (7-CKA) and 5,7-dichlorokynurenic acid (5,7-DCKA), are based on kynurenic acid derivatives (Dingledine et al., 1999; Kew and Kemp, 2005) Most glycine site competitive antagonists are not subtype specific (Table 1.1)

Table 1.1 Competitive NMDA receptor antagonists and their binding affinity to NR2 subunits.2

Ki values are determined from inhibition of currents from NMDA receptors expressed

in Xenopus oocytes

Abbreviations: 7-CKA, 7-chlorokynurenic acid; 5,7-DCKA, 5,7-dichlorokynurenic acid; CGP 61594, (±)-trans-4-[2-(4-azidophenyl)acetylamino]-5,7-dichloro-1,2,3,4-tetrahydroquinoline-2-carboxylic acid; NVP-AAM077, [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid; PMPA, (RS)-4-(phosphonomethyl)-piperazine-2-carboxylic acid; PPDA, (2S*,3R*)-1-(phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid; (R)-AP5, (R)-2-amino-5-phosphonopentanoate; (R)-AP7, (R)-2-amino-7-phosphonoheptanoate; (R)-CPP, (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid

2

Reprinted from Current Opinion in Pharmacology, Vol 7, P Paoletti, J Neyton, NMDA receptor

1.4.2.3 Uncompetitive antagonists

NMDA receptor activity can also be inhibited by occlusion of the ion channel pore which prevents ion influx Compounds acting through this mechanism are termed uncompetitive antagonists and they act on only activated receptors which have opened channel pores The bounded blockers will be trapped upon channel closure and reversal from the trapped blocked state is normally slow (Dingledine et al., 1999) The external Mg2+ ion is a classical native uncompetitive antagonist that acts in

a voltage-dependent manner Other examples of NMDA receptor open channel blockers include dizolcipine (MK801), dissociative anaesthetics phencyclidine (PCP), thienylcyclohexylpiperidine (TCP) and ketamine, and the clinically used drugs amantadine and memantine (Yamakura and Shimoji, 1999; Kew and Kemp, 2005; Paoletti and Neyton, 2007) Although most of the channel blockers, such as PCP and ketamine, do not show subtype selectivity, some of them, such as Mg2+ and MK801, have higher affinity towards NR2A and NR2B containing NMDA receptors (Yamakura and Shimoji, 1999; Paoletti and Neyton, 2007)

1.5 NMDA Receptors at the Glutamatergic Synapse

1.5.1 Structure of the glutamatergic synapse

Glutamatergic synapses play important roles in the brain physiology and pathophysiology (Zhuo, 2009) The establishment, maturation and activity-induced modification of the glutamatergic synapses can affect the efficacy of glutamatergic transmission, which forms the basis for development of neural circuits, proper brain functions and synaptic plasticity (Sheng and Hoogenraad, 2007; Nadif Kasri et al.,

2009) One of the key proteins that play a crucial role in synaptic transmission is the NMDA receptor which resides in the glutamatergic synapse (Zhuo, 2009)

Structurally, glutamatergic synapses reside on dendritic spines and consist of the presynaptic terminal and the postsynaptic terminal which sandwich the synaptic cleft (Kornau, 2009) (Figure 1.4) The release and diffusion of L-glutamate from the presynaptic terminals through the synaptic cleft to the postsynaptic terminals leading

to activation of NMDA receptors form the basis of glutamatergic synaptic transmission The postsynaptic density (PSD), found at the postsynaptic terminal where synaptic transmission occurs, is a protein network consisting of glutamate receptors and membrane proteins which are bound to the scaffolding protein PSD-95 (Okabe, 2007; Sheng and Hoogenraad, 2007) The PSD-95 protein organizes the receptors and many signalling proteins at the PSD and plays an important regulatory role in synaptic transmission (Kim and Sheng, 2004)

The postsynaptic membrane can be split into two regions: the synaptic region which contains the PSD, and the extrasynaptic region (Triller and Choquet, 2005) These regions are empirically defined by electrophysiologists and microscopists but pictorially, a region is extrasynaptic if it does not have the following: a synaptic cleft, vesicles containing neurotransmitters in a presynaptic active zone and a PSD (Sheng and Hoogenraad, 2007; Petralia et al., 2010) (Figure 1.4)

Figure 1.4 The glutamatergic synapse.3 The postsynaptic membrane can be divided into two segments, the synaptic region and the extrasynaptic region Both NR2A- and NR2B-containing NMDA receptors can be found in both locations At the synaptic region, a region called the PSD is critical for synaptic transmission At the PSD, the MAGUK family of scaffolding proteins such as SAP-102 and PSD-95 anchor glutamate receptors to the membrane surface and facilitate intracellular signalling pathways Figure was adapted to highlight the postsynaptic synapse consists of distinct regions such as the synaptic region and the extrasynaptic region

3

Adapted by permission from Macmillan Publishers Ltd: Nature Review Neuroscience, Lau CG, Zukin

RS (2007) NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders Nat Rev Neurosci 8:413-426., copyright 2007

Synaptic Region

Extrasynaptic Region

1.5.2 NMDA receptors residing at the postsynaptic membrane

1.5.2.1 Subunit composition

As the channel properties of NMDA receptors are highly dependent on the subunit composition, together with the differential expression of the NMDA receptor subunits, the subunit heterogeneity is believed to be critical during synaptic development, maturation and other forms of synaptic plasticity (van Zundert et al., 2004)

During synaptic development, synaptic NMDA receptors are predominantly NR2B-containing at the immature synapse and upon synapse maturation, synaptic NMDA receptors are found to be NR1/NR2B, NR1/NR2A and NR1/NR2A/NR2B receptors (Groc et al., 2009) In the mature synapse, NR2A-containing NMDA receptors are found at both synaptic and extrasynaptic sites, with a higher proportion

in the synaptic sites while NR2B-containing receptors are persistently found at both sites (Sheng and Hoogenraad, 2007; Petralia et al., 2010) These changes in composition of NMDA receptors within the postsynaptic membrane during development can lead to an alteration of NMDA receptor function, which is a critical determinant in synaptic transmission and synapse plasticity Therefore, mechanisms that can contribute to the changes in composition of NMDA receptors, such as differences in insertion (Barria and Malinow, 2002) or internalization (Roche et al., 2001; Lavezzari et al., 2003; Lavezzari et al., 2004) and/or lateral diffusion (Tovar and Westbrook, 2002; Groc et al., 2006; Newpher and Ehlers, 2008), are likely to be key parameters for the regulation of synaptic plasticity

1.5.2.2 Signaling mechanisms

The location of NMDA receptors may potentially affect the resulting intracellular signaling cascade upon NMDA receptor activation It was found that synaptic NMDA receptors are implicated in LTP induction while activation of extrasynaptic NMDA receptors induced long term depression (LTD) (Lu et al., 2001) Differential activation of NMDA receptors can also trigger either neuroprotection or cell death pathways It was found that activation of synaptic NMDA receptors induced cAMP response element binding protein (CREB) – dependent and brain-derived neurotrophic factor (BDNF) gene which activate an anti-apoptotic pathway (Hardingham et al., 2002; Vanhoutte and Bading, 2003) Conversely, extrasynaptic NMDA receptors activation blocked nuclear signaling to CREB, inhibited BDNF expression and induced mitochondrial dysfunction, eventually leading to cell death (Hardingham et al., 2002; Leveille et al., 2008) In addition, Xu and colleagues also reported that extrasynaptic activation led to calpain-induced p38 activation cell death (Xu et al., 2009)

1.6 Modulation of NMDA Receptors

NMDA receptors can be modulated in a variety of ways Allosteric modulators, found either endogenously or as synthetic molecules, can modulate NMDA receptor functions In addition, phosphorylation of the CTD of NMDA receptors or protease interaction can modify NMDA receptor properties

1.6.1 Allosteric modulators

1.6.1.1 Protons

Extracellular protons can inhibit NMDA receptors in a non-competitive and voltage-independent manner (Giffard et al., 1990; Traynelis and Cull-Candy, 1990; Vyklicky et al., 1990) Proton sensitivity is controlled by exon 5 in the NR1 subunit and the identity of the NR2 subunit (Traynelis et al., 1995) NMDA receptors that contain NR1a subunits, i.e absence of exon 5, can be inhibited by protons with IC50values of pH 7.2-7.4 (Traynelis et al., 1995) Presence of exon 5 in NR1 relieved pH inhibition for NR2A, NR2B and NR2D containing receptors (IC50 of pH 6.6-6.8) while NR2C containing receptors are not sensitive to protons regardless of the splice form of NR1 (IC50 of pH 6.2) (Traynelis et al., 1995) Hence, it has been suggested that NMDA receptors are tonically inhibited under normal physiological conditions and this property can help to inhibit NMDA receptors and reduce neurotoxicity during pathologies such as ischemia or seizure where the extracellular environment is mildly acidic in nature (Tang et al., 1990; Traynelis et al., 1995; Dingledine et al., 1999)

Proton inhibition has an IC50 of about pH 7.4 or a concentration of approximately 50nM and hence, under physiological conditions, extracellular protons should tonically inhibit about 50% of native neuronal NMDA receptors (Traynelis et al., 1995; Yamakura and Shimoji, 1999)

Although the exact location of the proton sensor is unknown, mutagenesis studies suggest that molecular determinants of proton sensitivity are reported to be located at the extracellular ends of M3 and the linkers connecting S2 to M3 and M4 (Low et al., 2003) As these regions are intimately connected to the activation gate of NMDA receptors, the proton sensor is proposed to be closely coupled to the channel pore movement (Chang and Kuo, 2008; Mony et al., 2009a)

One common downstream mechanism engaged by allosteric modulators of the NMDA receptors is the modification of proton sensitivity A modulator-induced subtle shift in the pKa can alter the tonic proton inhibition at physiological pH: enhanced proton sensitivity will lead to inhibition of the receptors while reduced proton sensitivity will lead to receptor potentiation

1.6.1.2 Zinc ions (Zn 2+ )

Zinc ions are found extracellularly at excitatory synapses in the brain and they can target and modulate NMDA receptors (Paoletti et al., 2009) Zn2+ can inhibit NR2A-containing NMDA receptors with apparently two distinct binding profiles: a high affinity voltage-independent inhibition and a second low affinity voltage-dependent inhibition (Williams, 1996; Chen et al., 1997) The molecular determinants

of high affinity Zn2+ binding have been mapped to the ATD of NR2A subunit and

Zn2+ can inhibit NR2A-containing NMDA receptors at low nanomolar concentrations (IC50 ~ 15nM) in a non-competitive, voltage-independent manner (Chen et al., 1997; Paoletti et al., 1997; Choi and Lipton, 1999; Fayyazuddin et al., 2000; Low et al., 2000; Paoletti et al., 2000) In addition, Zn2+ can also bind to the ATD of NR2B subunit, but not NR2C or NR2D subunits but with a lower affinity compared to NR2A (Rachline et al., 2005; Karakas et al., 2009) This binding of Zn2+ to NR2B can inhibit NR1/NR2B receptors in a voltage-independent manner with an IC50 of about

1 M (Williams, 1996; Chen et al., 1997; Rachline et al., 2005) Hence, Zn2+ can selectively inhibit NR2A-containing receptors at nanomolar concentrations Mechanistically, it has been proposed that the high affinity Zn2+ inhibition of NR1/NR2A receptors is due to modification of proton sensitivity and involves enhancement of the tonic proton inhibition (Choi and Lipton, 1999; Low et al., 2000; Erreger and Traynelis, 2008)

1.6.1.3 Polyamines

Spermine, spermidine and putrescine belong to the family of polyamines and are found endogenously in the body (Rock and Macdonald, 1995) They can be released into the extracellular space in the CNS upon neuronal stimulation and have been reported to interact with the NMDA receptors (Rock and Macdonald, 1995) Recently, the binding site of spermine has been mapped to the ATD of NR1, NR2A and NR2B (Han et al., 2008)

Spermine and spermidine can modulate NMDA receptor function in three ways Firstly, spermine can function as a voltage-dependent block by entering the channel pore inhibit ion influx (Rock and MacDonald, 1992) Secondly, at low glycine concentrations, spermine can potentiate NMDA receptor responses by increasing glycine affinity (glycine-dependent potentiation) (McGurk et al., 1990) Glycine-dependent potentiation can only be observed in NR2A- and NR2B- containing receptors but not NR2C- and NR2D-containing receptors and is not affected by the splice forms of NR1 subunits (Williams et al., 1994; Williams et al., 1995) Lastly, under saturating glycine conditions, spermine potentiates NMDA receptor function in a voltage- and glycine- independent manner (McGurk et al., 1990; Benveniste and Mayer, 1993) This form of potentiation can only be observed in NR2B-containing receptors (Williams, 1994; Zhang et al., 1994; Traynelis et al., 1995) It was found that at physiological pH, spermine shifts the pKa of the proton sensor to a more acidic level, leading to the relief of the tonic proton inhibition due to reduced proton sensitivity, which results in the potentiation of NR2B-containing receptors (Traynelis et al., 1995)

1.6.1.4 NR2B-selective allosteric antagonists

Ifenprodil and its family of phenylethanolamines are a class of synthetic NMDA receptor antagonists that selectively inhibits NR2B-containing NMDA receptors (Williams, 1993; Chenard and Menniti, 1999) It is generally accepted that ifenprodil binds to the ATD of the NR2B subunit, although molecular determinants of ifenprodil binding have been found in the ATD of NR1 subunit as well (Gallagher et al., 1996; Masuko et al., 1999; Perin-Dureau et al., 2002; Wong et al., 2005; Madry et al., 2007a; Ng et al., 2007; Han et al., 2008) Ifenprodil inhibits NR1/NR2B receptors

in a non-competitive and voltage-independent manner with an IC50 of 0.34 M and has more than 400-fold affinity for NR1/NR2B receptors compared to NR1/NR2A receptors (Carter et al., 1988; Williams, 1993) Ifenprodil inhibition of NR1/NR2B receptors is dependent on pH, with low inhibition at alkaline pH (Pahk and Williams, 1997) The mechanism of ifenprodil inhibition is similar to Zn2+ whereby ifenprodil enhances proton sensitivity by shifting the pKa of the proton sensor to a more alkaline level, thus resulting in more receptors being inhibited at physiological pH (Mott et al., 1998)

ATD-targeted allosteric antagonists are usually partial antagonists, i.e saturating antagonists concentration will not lead to the full inhibition of receptors (Paoletti and Neyton, 2007) Hence, it is not surprising that ifenprodil inhibits NR2B-containing receptors with a maximum inhibition of about 90% (Mott et al., 1998) Nonetheless, these characteristics of allosteric inhibitors are a therapeutic advantage

in circumstances where residual NMDA receptor activity might be necessary for proper brain function (Paoletti and Neyton, 2007) Hence, NR2B-selective antagonists have received a lot of attention as potential therapeutic drugs for many brain disorders (Mony et al., 2009a)