Investigating the Role of the Pre-M1 Helix and Other Highly Conserved Regions in NMDA Receptor Function Open Access
McDaniel, Miranda (Fall 2019)
Abstract
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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