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«ROLE OF INTERDOMAIN AND INTERSUBUNIT CONTACTS FOR NMDA RECEPTOR FUNCTION Vom Fachbereich Biologie der Technischen Universität Darmstadt zur ...»

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ROLE OF INTERDOMAIN AND INTERSUBUNIT CONTACTS

FOR NMDA RECEPTOR FUNCTION

Vom Fachbereich Biologie der Technischen Universität Darmstadt

zur

Erlangung des akademischen Grades eines Doctor rerum naturalium

genehmigte Dissertation von

Dipl Bioinf. Ceyhun Tamer

aus Offenbach am Main

Berichterstatter (1. Referent): Prof.Dr. Bodo Laube

Mitberichterstatter (2. Referent): Prof.Dr. Kay Hamacher

Tag der Einreichung: 29. Juni 2012 Tag der mündlichen Prüfung: 31. August 2012 Darmstadt 2012 (D 17) „Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time.“ Thomas A. Edison Table of contents 3 Chapter 1 – General introduction 6 Ionotropic receptors mediate cell-cell communication 6 Ligand-gated ion channels (LGICs), a subtype of ionotropic receptors 7 Ionotropic glutamate receptors (iGluRs) 9 Modular composition of iGluRs 10 Overall organization of iGluR structure 13 Activation mechanism of iGluRs 18 Aim of this work 21 References 22 Chapter 2 – In silico approach to determine the molecular action of ligand binding to NMDA receptor subunits 27

Abstract

27 Introduction 28 Experimental procedures 32 Results 34 Discussion 41 Conclusions 43 References 44 Chapter 3 – Role of heterodimer interface interactions in the ligand binding domain for NMDA receptor function 47 Abstract 47 Introduction 48 Experimental procedures 50 Results 52 Discussion 63

–  –  –

Ionotropic receptors mediate cell-cell communication Communication between cells is generally achieved via signal molecules or ions. Nerve cells are specialized cells that contact other cells through so-called ‚synapses’. A synapse is formed by a presynaptic terminal where signal molecules, i.e. neurotransmitters, are released and a post-synaptic terminal where the neurotransmitters bind to specialized, membrane-bound proteins. These proteins are classified according to their respective biochemical reaction, i) metabotropic receptors, which induce secondary signal mechanisms that trigger a range of intracellular events, and ii) ionotropic receptors, which, upon ligand binding, open a channel that allows ions such as Na+, K+ or Cl- to flow along their electrochemical gradient. In contrast, so-called ion channels are proteins that are activated e.g. by voltage changes, pH changes or mechanical stretch. Ion channels and ionotropic receptors are membrane-associated, oligomeric proteins and the ion channel pore is located in the membrane-domain. But only ionotropic receptors have an extracellular domain (ECD) where ligands can bind. Binding of a ligand to the ECD induces rearrangements in the protein that lead to the opening of the inherent ion channel (Mayer, 2006). Such receptors are involved in fast signal transmission as their operating time scale is in the range of a few milliseconds compared to seconds or minutes in metabotropic receptors (Kandel, 2000). Ionotropic receptors or their evolutionary ancestor proteins can be found throughout all life forms, e.g. bacteria, plants, insects, fish, birds, amphibians, reptiles and mammals. Their significance for cell-cell communication becomes apparent when mutations in the respective genes cause so-called channelopathies. Channelopathies are disorders or pathophysiological conditions that are caused by a mutation in an ion channel or ionotropic receptor. Examples for channelopathies caused by mutations in ionotropic receptors are, hyperekplexia or startle disease (Shiang et al., 1993), different forms of epilepsy (Steinlein et al., 1995, De Fusco et al., 2000, Baulac et al., 2001, Wallace et al., 2001, Endele et al., 2010), congenital myasthenic syndrome (Engel et al.,

1993) or mental retardation (Endele et al., 2010). Additionally, ionotropic receptors are implicated in numerous neurological diseases such as, Alzheimer’s, Parkinson’s or Huntington’s (Dingledine et al., 1999, Wu et al., 2006). In order to develop treatment for such disorders it is essential to gain insight into the structure and function of these proteins.

Ligand-gated ion channels (LGICs), a subtype of ionotropic receptors LGICs belong to the class of ionotropic receptors and are comprised of three superfamilies, the pentameric Cys-loop receptors, the tetrameric ionotropic glutamate receptors (iGluRs) and the trimeric ATP-gated channels (P2X receptors). Receptors are generally classified according to a specific ligand that activates the respective receptor type. However, all these receptors have a common basic structure of an extracellular domain (ECD), a transmembrane domain (TMD) and an intraextracellular C-terminal domain (CTD). Families within the Cys-loop receptor superfamily are nicotinic acetylcholine receptors (nAchRs), glycine receptors (GlyRs), γ-amino butyric acid type A receptors (GABAARs) and serotonin type 3 receptors (5HT3 receptors). These receptors form pentameric complexes, the binding pocket for ligands is located in the interface between the ECDs of two neighboring subunits, the transmembrane domain of each subunit is formed by four transmembrane helices (TM1-TM4) and the TM2s in the pentamer are lining the ion channel pore and the CTD lies extracellular. Interestingly, nAChRs and 5-HT3 receptors are cationic channels and lead to depolarization of the post-synaptic potential, whereas GlyRs and GABAARs are anionic channels and lead to hyperpolarization. Cys-loop receptors represent an interesting pharmacological target as anesthetics, steroids and benzodiazepines act via modulation of GABAA or glycine receptor function (Thurmon et al., 1996a, Maksay et al., 2001, Thio et al., 2003, Garcia et al., 2010).





The N-methyl-D-aspartate receptors (NMDARs), (RS)-2-amino-3-(3-hydroxy-5methyl-4-isoxazolyl)propionic acid receptors (AMPARs) and kainate receptors (KARs) form three distinct subfamilies in the family of iGluRs. Commonly, iGluRs can be activated by glutamate, however, the efficiency of activation depends on the subunit composition of the receptor (Dingledine et al., 1999). In contrast to Cys-loop receptors, the respective subunits assemble to form tetrameric complexes (Sobolevsky et al., 2009). Ligand-binding occurs in the ECD but not between, but within subunits. The TMD is organized by three transmembrane domains (TM1, TM3 and TM4), the M2 domain is a re-rentrant loop that is not spanning the membrane.

The CTD is located on the intracellular side of the membrane (Madden, 2002).

IGluRs convey the majority of excitatory neurotransmission in the mammalian central nervous system and as such they are also implicated in the glutamate-induced excitotoxicity (Dingledine et al., 1999). Excessive activation of iGluRs by glutamate leads to overexcitation of the cell, which drives the cell into apoptosis. This basic principle underlies many neurodegenerative disorders, e.g. Alzheimer’s, Parkinson’s, Huntington’s and multiple sclerosis (Dingledine et al., 1999).

Fig. 1. Overview of the three superfamilies in LGICs. Cartoon representation of examples for Cys-loop receptors, iGluRs and P2X receptors. Each subunit is colored differently for a

better overview. (A-B) Side/Top view of the pentameric GluCl crystal structure (PDB ID:

3RHW, Hibbs et al., 2011), which is an invertebrate Cys-loop receptor and is depicted here as a model for vertebrate Cys-loop receptors, (C-D) Crystal structure of the tetrameric GluA2-type AMPA receptor (PDB ID: 3KG2, Sobolevsky et al., 2009), (E-F) Crystal structure of the trimeric ATP-gated P2X4 receptor (PDB ID: 3I5D, Kawate et al., 2009).

LBD ligand-binding domain, NTD N-terminal domain, TMD transmembrane domain.

ATP-gated P2X receptors are cationic channels and have only two transmembrane domains (TM1, TM2), the N- and C-termini are intracellular located and the extracellular loop between TM1 and TM2 of two subunits form the ATPbinding site (Kawate et al., 2009). In contrast to Cys-loop and iGluRs, the P2X receptors form trimeric complexes. P2X receptors are implicated in a variety of physiological processes, such as synaptic transmission, inflammation, sensing of taste and pain (Kawate et al., 2009).

Although, all the ligand-gated ion channels serve the function of ligandinducible ion channels that conduct ions along their respective electrochemical gradient, the molecular structure and overall organization of the receptors differ greatly. In the future it will be interesting to compare the structure and function relationship between the superfamilies in order to identify basic principles of receptor function. However, first the structure and function relationship within the superfamilies need to be analyzed. The work presented here is focused on the research of structure and function relationship in NMDARs and iGluRs in general.

Ionotropic glutamate receptors (iGluRs) Based on distinct functional properties iGluRs are further discriminated into non-NMDA receptors, i.e. AMPA and kainate receptors, and NMDA receptors (Dingledine et al., 1999). Four AMPA receptor subunits exist, which are GluA1-4 and each subunit has two isoforms, i.e. flip or flop (Hollmann and Heinemann, 1994). For kainate receptors five subunits are known, i.e. GluK1-5 (Hollmann and Heinemann, 1994). Non-NMDA receptors are non-selective cationic channels. Thus, Na+ and K+ permeate through the ion channel pore, however, Ca2+ permeability depends on the subunit composition (Traynelis et al., 2010). AMPARs lacking the GluA2 subunit will be permeable for Ca2+, but if a GluA2 subunit is incorporated it depends on posttranscriptional RNA editing at the so-called Q/R editing site, if the receptor is Ca2+ permeable or not (Traynelis et al., 2010). Non-NMDA receptors are mainly responsible for the fast depolarization of the post-synaptic membrane.

Subunits of the NMDA receptors are, GluN1 subunits with 8 isoforms, the GluN2A-D and GluN3A-B subunits (Monyer et al., 1994, Dingledine et al., 1999, CullCandy et al., 2001). NMDA receptors are obligate heteromeric complexes, the ‘conventional’ NMDA receptor is composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits (Laube et al., 1998, Rosenmund et al., 1998, Dingledine et al., 1999, Furukawa et al., 2005). In comparison to AMPA and kainate receptors, the conventional NMDA receptors do not only conduct Na+ and K+ but are highly Ca2+ permeable (Mayer and Westbrook, 1987, Burnashev et al., 1995).

Also, at resting potential conventional NMDA receptors are blocked by extracellular Mg2+, which functions as a channel blocker (Dingledine et al., 1999). When the postsynaptic membrane is depolarized by non-NMDA receptors, Mg2+ ions are repelled from the NMDAR channel pore and NMDA receptors can be activated. Thus, conventional NMDA receptors become only active upon ligand-binding and membrane depolarization, therefore they are also referred to as coincidence detectors (Dingledine et al., 1999). The high Ca2+ permeability is important as Ca2+ triggers second messenger signal cascades inside the cell, which are required for learning and memory formation (Collingridge and Bliss, 1995, Yashiro and Philpot, 2008).

NMDA receptors composed of glycine binding GluN1 and GluN3 subunits are referred to as ‘excitatory glycine receptors’ and exhibit very small currents (Chatterton et al., 2002, Madry et al., 2007a, Awobuluyi et al., 2007). Although these receptors have not been identified in vivo yet, they pose a very interesting target for the study of structure and function relationship as receptor currents can be potentiated up to ~125-fold by the co-application of glycine, Zn2+ and a GluN1 antagonist (Madry et al., 2008, Madry et al., 2010).

NMDARs stand out in the iGluR family because of their obligate heteromeric assembly, ligand- and voltage-dependency, implication in neurological disorders and the high degree of receptor modulation (Dingledine et al., 1999, Madry et al., 2008, Traynelis et al., 2010). Thus, understanding structure and function relationship in NMDA receptors is a great and exciting challenge.

Modular composition of iGluRs It is currently believed that iGluRs have evolved from bacterial ancestor proteins (Lampinen et al., 1998, Masuko et al., 1999, Chen et al., 1999, Wollmuth et al., 2004, Matsuda et al., 2005). This hypothesis was supported by the identification of a missing link, the glutamate-binding GluR0 subtype receptors, which show homologies to prokaryotic potassium channels and eukaryotic iGluRs. (Chen et al., 1999, Kuner et al., 2003). This evolutionary connection was further supported by the identification of amino acid binding proteins, which show homologies to different domains in the modular composition of iGluRs.

–  –  –

The most distal part of an iGluR subunit from the membrane is the N-terminal domain (NTD). This domain shows sequence homology to the bacterial periplasmic Leucine-Isoleucine-Valine binding protein (LIVBP) and consists of ~400 amino acids (Masuko et al., 1999, Matsuda et al., 2005). The NTD has a bilobed structure, which means that the NTD is subdivided into the R1 and R2 domains. At the interface between these two subdomains a cavity is formed that is also a binding-site for allosteric modulators, e.g. Zn2, which has been shown to inhibit GluN1/GluN2A receptor currents (Paoletti et al., 1997, Herin and Aizenman, 2004). The R1 and R2 subdomains are in constant movement in vivo opening and closing the binding-site periodically. This movement has been termed ‘oscillation’ and the frequencies of these oscillations are subunit specific and largely affect desensitization and channel open probability (P0) of GluN1/GluN2 receptors (Yuan et al., 2009, Gielen et al., 2009). Structure and function studies with AMPA receptors have implicated that the NTDs determine the subunit specific assembly. In detail, GluA1 and GluA3 subunits as well as GluA2 and GluA4 dimerize readily whereas GluA1 and GluA2 or GluA2 and GluA3 subunits do not (Ayalon and Stern-Bach, 2001, Ayalon and Stern-Bach, 2005, Rossmann et al., 2011). However, if the NTDs are removed the subunits dimerize with no favored combination (Ayalon and Stern-Bach, 2001, Pasternack et al., 2002). Taken together, NTDs are implicated in the assembly of iGluRs and the modulation of iGluR function. However, little is known about the functional roles of the GluN3 NTDs.



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