Regulation of AMPA receptor function and synaptic localization by stargazin and PSD-95

The majority of excitatory transmission in the brain is mediated by
glutamatergic synapses. Rapid synaptic signaling is mediated by
AMPA and kainate receptors, whereas NMDA receptors mediate slow
synaptic currents. Pathophysiological activation of glutamatergic
neurons can lead to excitotoxicity and neuronal death, for example
in ischaemia and neurodegenerative disorders. Therefore, studying
the structure and function of AMPA receptors is important for
understanding general mechanisms of synaptic transmission as well
as for the development of new therapies. AMPA receptors are
associated with auxiliary subunits called Transmembrane AMPA
Receptor Regulatory Proteins (TARPs). The first identified member
of this family was stargazin. Given the structural similarity to
the γ1 subunit of skeletal muscle voltage-gated Ca2+channels,
stargazin is also called γ2. The stargazer mouse is a spontaneous
mutant that lacks AMPA receptors in granule cells of cerebellum and
suffers from ataxia. In addition to stargazin, the family includes
γ3, γ4 and γ8. TARPs regulate all aspects of AMPA receptor function
- from early steps of synthesis and trafficking to the cell
surface, to synaptic localization and biophysical properties. TARPs
interact with PSD-95, a main scaffolding protein of excitatory
synapses that belongs to the Membrane-Associated Guanylate Kinases
(MAGUK) family. Via this interaction AMPA receptors are localized
to the synapse. PSD-95 clusters many other synaptic proteins and
organizes signaling complexes in the synapse. The goal of this
thesis was to investigate the role of stargazin in regulating the
antagonism of AMPA receptors. I focused on the commonly used
antagonists CNQX, GYKI-53655 (GYKI) and CP-465,022 (CP) and
explored how stargazin changes the inhibition of AMPA receptors by
these drugs. The second goal was to assess the role of PSD-95 in
synaptic function. More specifically, I aimed to investigate how an
increased level of PSD-95 in a neuron affects AMPA and NMDA
currents, as well as the presynaptic function of a neuron. In the
first part of my thesis I used the heterologous Xenopus oocyte
expression system to express AMPA receptor subunits alone or with
stargazin. Using the two-electrode voltage clamp, I measured the
glutamate-evoked currents and obtained dose-response curves for
CNQX, GYKI and CP. I found that stargazin decreases the affinity of
GluR1 for CNQX, which was explained by the partial agonistic effect
of CNQX in the presence of stargazin. In contrast, stargazin
increases the affinity for GYKI, and has only a small effect on CP.
I also tested the effect of stargazin on recently described
GYKI-insensitive receptors and found that inhibition of these
receptors is restored by co-expression with stargazin. My data
strongly suggest that the identified residues do not constitute the
full GYKI-binding site. I could also show that the ectodomain of
stargazin controls the changes in antagonist sensitivity of the
receptors. In the second part of my thesis I used cultured
hippocampal slices and Semliki Forest virus to overexpress
PSD-95:GFP in CA1 region of hippocampus. I recorded simultaneously
from a cell overexpressing PSD-95 and a neighboring control cell
and compared their AMPA and NMDA currents. I confirmed the finding
that overexpression of PSD-95 robustly increases currents mediated
by AMPA receptors. In contrast to other studies, I observed that
PSD-95 increases NMDA currents, although to smaller extent. I
addressed the debated role of PSD-95 in regulating the presynatic
release probability and found that overexpression of PSD-95 did not
change glutamate release probability. Importantly, I observed that
cells overexpressing PSD-95 have a lower rectification index of
synaptic AMPA receptors, strongly suggesting that PSD-95
overexpression led to an increased fraction of AMPA receptors that
lack GluR2 subunit. In conclusion, the work presented in this
thesis gives further insights into AMPA receptor physiology, both
from the aspect of pharmacology and synaptic trafficking. The
results of co-expression of stargazin with the previously described
GYKI-insensitive GluR1 mutants strongly indicate that TARP
interacts with the linker domains of AMPA receptors. This finding
is of great importance for understanding the molecular mechanism of
AMPA-TARP interaction. Furthermore, this thesis shows that PSD-95
regulates both AMPA and NMDA synaptic currents by increasing the
number of synaptic receptors. In addition, my data suggest that
PSD-95 enriches the number of GluR2-lacking receptors in the
synapse. Given the Ca2+permeability of GluR2-lacking receptors and
their implication in plasticity and excitotoxicity, this finding is
important for understanding how the synaptic localization of these
receptors is regulated.