Investigating the Role of the Pre-M1 Helix and Other Highly Conserved Regions in NMDA Receptor Function Restricted; Files Only

McDaniel, Miranda (Fall 2019)

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NMDA receptors (NMDARs) are calcium-permeable ion channels with a critical role in the slow-component of fast excitatory neurotransmission. Typically comprised of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits, these receptors respond to the synaptic release of glutamate to facilitate the opening of the cation-selective pore. The GluN2 subunits can be one of four subtypes (A-D), while the GluN1 subunits can be one of eight splice variants. Moreover, each NMDAR subunit contains four semi-autonomous domains—an amino terminal domain (ATD), an agonist-binding domain (ABD), a transmembrane domain (TMD), and a carboxy terminal domain (CTD)— connected through a series of flexible linkers. In the healthy brain, NMDARs are critical for neuronal development, learning and memory, synaptic plasticity, and several other important processes. Pathologically, they have been associated with such neurological disorders as Parkinson’s disease, Alzheimer’s disease, and schizophrenia. As we learn more about their role in brain physiology and pathology, it becomes increasingly important to develop a more comprehensive understanding of NMDAR function. In this dissertation, I explore the functional role of three highly conserved regions of the NMDAR: the pre-M1 helix, the GluN2 glutamate binding site, and the GluN1 exon 5 motif. Guided by genetic regulation and variation, I introduced purposeful mutations within these regions and report the effect of these substitutions on a multitude of channel properties. I show that these regions are likely under such strict genetic protection as a consequence of their considerable influence on receptor function. Moreover, I present a structure-based model to investigate how independent subunits contribute to the activation of NMDARs. Finally, I show that deactivation rate correlates with both proton sensitivity and glutamate egress time. Together, the results presented in this dissertation shed light on the role of specific highly conserved regions— and the amino acids therein—in the function of the NMDAR ion channel. Moreover, this study emphasizes the value of using genetics to guide the rational design of investigative scientific inquiry.

Table of Contents

Chapter 1: Introduction. 1

Neurotransmission and Cognition. 1

Excitatory Neurotransmission Involves Ionotrobic and Metabotropic Glutamate Receptors 3

Ionotropic Glutamate Receptors are Involved in Fast Excitatory Neurotransmission. 4

NMDA Receptors 6

NMDA Receptor Deactivation is Defined by Four Receptor Properties 7

The NMDA Receptor is Involved in Brain Physiology and Pathology. 9

The Properties of the Tetrameric NMDA Receptors Are Dependent on Subunit Composition. 11

Triheteromeric NMDA Receptors Have Unique Functional Properties 19

NMDA Receptor Gating. 20

The Pre-M1 Helix Controls Channel Gating. 21

NMDAR Gating is Thought to Involve a Triad Between the TMD and Linkers 23

Pre-M1 Helices are Highly Conserved. 26

Summary. 31

Chapter 2: Materials and Methods. 33

DNA Constructs 33

Site-Directed Mutagenesis 33

Two electrode voltage clamp recordings from Xenopus oocytes 37

MTSEA Assay. 38

HEK Cell Culture 39

Β-Lactamase Trafficking Assay. 39

Whole cell current recordings from transfected HEK cells 40

Homology Modeling, Molecular Dynamics, and Energy Change Calculations 42

Fitting of Structure-Based Mechanisms to Macroscopic Current Responses 44

Statistical Analysis 45

Chapter 3: NMDA Receptor Channel Gating Control by the Pre-M1 Helix*. 46

Abstract 47

Introduction. 48

Results 52

Effects of Pre-M1 Mutations on Agonist Potency and Response Time Course 52

. 57

Pre-M1 Mutations Alter NMDA Receptor Single Channel Properties 59

Subunit-Specific Pre-M1 Helix Interactions 68

Discussion. 73

Chapter 4: Gating Effects of Disease-Associated Mutations within the Pre-M1 Helix*. 80

Abstract 81

Introduction. 82

Results 87

Functional Consequences of Various Amino Acids 87

. 89

Effects of the Pre-M1 Proline Mutation in Triheteromeric Receptors 91

Designing a Structure Based Model to Elucidate NMDAR Gating Mechanisms 95

Discussion. 101

Chapter 5: Functional Effects of a Disease-Associated Mutation in the S1-M1 Linker 108

Abstract 109

Introduction. 110

Results 113

GluN1-L551P Increases Agonist Potency. 113

GluN1-L551P Alters Receptor Pharmacology. 114

GluN1-L551P Slows Receptor Deactivation. 117

GluN1-L551P Reduces Surface Expression. 119

Discussion. 122

Chapter 6: Functional Effects of Genetic Variation within NMDA Receptor Domains. 126

Abstract 127

Introduction. 128

Results 131

Structure of the GluN1 Exon 5 Motif Controls Deactivation Rates 131

. 135

Exon 5 Mediated Changes in Receptor Deactivation Correlates with Proton Sensitivity. 137

Mutations Within GluN2B ABD Accelerate Deactivation Rate. 137

Deactivation Rate Correlates with Ligand Egress time for GluN2B ABD Mutations 139

Discussion. 142

Chapter 7: Discussion. 148

The Pre-M1 Helix Controls Channel Gating. 149

The Residues of the Pre-M1 Helix Contribute to Normal NMDA Receptor Function. 150

The Pre-M1 Helix Contributes to Subunit Specific Contributions to Receptor Function. 151

The Pre-M1 Helix is Part of an Aromatic Gating Network. 153

Mechanism of Pre-M1 Control of Gating. 156

Modeling the NMDA Receptor Gating Mechanism.. 158

Pre-Gating of Only Three Subunits is Required for Channel Opening. 158

A Structure-Based Model Can Predict Conformational Transitions in Individual Subunits 160

A Disease-Associated Mutation in the S1-M1 Linker Impairs Receptor Function. 161

GluN1-L551P Alters Receptor Pharmacology, Kinetics, and Surface Expression. 162

The Functional Consequences of Glun1-L551P are Consistent with an Abnormal Phenotype. 164

The Effects on Deactivation Rate of Highly Conserved Regions of the NMDA Receptor. 165

Exon 5: Deactivation Rate Correlates with pH Sensitivity. 165

Agonist Binding Domain: Deactivation Rate Correlates with Glutamate Egress 167

Future Directions 168

Conclusion. 170

Chapter 8: References 171

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