Mechanisms of GluN2D subunit-specific control of synaptic signaling Open Access

Vance, Katie Marie (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/gt54kn28r?locale=en
Published

Abstract



NMDA receptors are members of a class of ionotropic glutamate receptors that also
includes AMPA, kainate, and delta receptors. NMDA receptors mediate the slow
component of excitatory synaptic transmission in the central nervous system and have a
role in learning, memory, and neuronal development. Two glycine-binding GluN1
subunits assemble with two glutamate-binding GluN2 subunits to form a functional
NMDA receptor, while the four GluN2 subunits (GluN2A-D) control a majority of the
properties of the receptor. GluN2D-containing NMDA receptors have an unusually slow
deactivation time course following the removal of L-glutamate and low channel open
probability compared to the other GluN2 subunits. This dissertation focuses on the
molecular mechanisms that control the key properties of GluN1/GluN2D NMDA
receptors and how these properties contribute to the synaptic activity of the subthalamic
nucleus. The data presented here show that the deactivation time course of
GluN1/GluN2D NMDA receptors is ligand-dependent, with L-glutamate causing a
slower deactivation time course than any other linear ligand evaluated. RNA splicing of
the GluN1 amino-terminal domain also controls the deactivation time course, agonist
EC50, and channel open probability of GluN1/GluN2D receptors. A gating scheme of
NMDA receptor activation is presented that describes the key characteristics of
GluN1/GluN2D NMDA receptor gating as well as identifies specific rate constants
controlled by the GluN1 amino-terminal domain. Finally, the data presented here suggest

that GluN2D-containing NMDA receptors contribute to the excitatory postsynaptic
currents of the subthalamic nucleus.

Table of Contents

Table of Contents

Chapter 1: Background
1
1.1. Abstract
1
1.2. Introduction to NMDA receptors
2
1.3. NMDA receptor structure
4
a. Subunit organization and stoichiometry
4
b. Amino-terminal domain
11
c. Ligand-binding domain
12
1.4. NMDA receptor pharmacology
18
a. NMDA receptor agonists
18
b. NMDA receptor competitive antagonists
22
c. NMDA receptor noncompetitive antagonists
24
d. NMDA receptor uncompetitive antagonists
28
e. NMDA receptor allosteric potentiators
30
1.5. NMDA receptor channel activation and gating
30
a. Functional features of NMDA receptor activation
30
b. Conceptual models of NMDA receptor gating
35
1.6. Neuronal GluN2D-containing NMDA receptors
39


Chapter 2: Methods
43
2.1. Molecular biology
43
2.2. Two-electrode voltage clamp recordings
43
2.3. Cell culture
44
2.4. Electrophysiology
44
2.5. Single channel analysis
45
2.6. Analysis of macroscopic recordings and concentration-response curves
48
2.7. Patch clamp recording from neurons in thin slices
52
2.8. Cerebellar granule cell culture
54
2.9. Statistical analysis
54


Chapter 3: NMDA receptor agonist pharmacology
56
3.1. Abstract
56
3.2. Introduction
57
3.3. Results
58
a. GluN1/GluN2D deactivation time course is ligand-dependent
58
b. The relationship between GluN1/GluN2D deactivation rate and agonist 65
potency
c. Molecular correlates of the LBD control of GluN1/GluN2D
69
deactivation time course
3.4. Discussion
81


Chapter 4: GluN1 splice variant control of GluN2D-containing NMDA receptors
85
4.1. Abstract
85
4.2. Introduction
86
4.3. Results
88
a. Exon 5 of the GluN1 ATD decreases agonist potencies
88
b. GluN1 splice variant control of GluN2D deactivation time course
89

c. Lys211 in the GluN1-1b ATD is necessary for exon 5 control of
95
potency and deactivation time course
d. Exon 5 increases the open probability of GluN2D-containing NMDA
97
receptors
e. Exon 5 does not influence GluN1/GluN2D conductance levels
106
f. The properties of GluN2D-containing NMDA receptors are conserved 109
in cell-attached patches
g. GluN1/GluN2D exhibit brief periods of high open probability
114
4.4. Discussion
116


Chapter 5: GluN1/GluN2D gating and channel activation
120
5.1. Abstract
120
5.2. Introduction
121
5.3. Results
122
a. Previously published NMDA receptor gating models cannot predict the 122
single channel and macroscopic characteristics of GluN2D-containing
NMDA receptors
b. A model with two parallel interconnected arms best describes the
129
single channel and macroscopic characteristics of GluN1/GluN2D
NMDA receptors
c. Scheme 5 identifies specific gating steps controlled by GluN1 exon 5
143
5.4. Discussion
145


Chapter 6: GluN1/GluN2D control of the synaptic activity of the subthalamic
148
nucleus
6.1. Abstract
148
6.2. Introduction
149
6.3. Results
153
a. Positive allosteric modulation of GluN1/GluN2D receptors expressed
153
in HEK 293 cells
b. Negative allosteric NMDA receptor modulation by dihydroquinilone-
157
pyrazolines
c. GluN2B and GluN2D-containing NMDA receptors in the STN are
163
inhibited or potentiated by subunit-selective allosteric modulators
d. GluN2D and GluN2B subunits contribute to evoked EPSCs in the STN 168
6.4. Discussion
180


Chapter 7: Discussion and Conclusion
185
7.1. Summary
185
7.2. GluN1/GluN2D deactivation time course is ligand-dependent
187
7.3. Splice variant control over GluN1/GluN2D NMDA receptor function
192
7.4. A two-arm linear model best predicts GluN1/GluN2D activation
196
7.5. GluN2D receptors contribute to the synaptic activity of the subthalamic
198
nucleus
7.6. Conclusion
208


Chapter 8: References
200

Document Outline
  • Vance Table of Contents 07162012
  • Vance Dissertation 07162012

About this Dissertation

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