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

摘要

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.