Mechanisms of GluN2D subunit-specific control of synaptic signaling Público
Vance, Katie Marie (2012)
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
- Vance Table of Contents 07162012
- Vance Dissertation 07162012
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