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

TRESK channel contribution to nociceptive sensory neurons excitability: modulation by nerve injury

Background: Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K(+) channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K(2P) channels after peripheral axotomy in mammals. Results: Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo. Conclusions: In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability

TRESK channel contribution to nociceptive sensory neurons excitability: modulation by nerve injury

<p>Abstract</p> <p>Background</p> <p>Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K⁺channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K_2Pchannels after peripheral axotomy in mammals.</p> <p>Results</p> <p>Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (<it>in vitro </it>axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured <it>in vivo</it>. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo.</p> <p>Conclusions</p> <p>In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability.</p

AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER). CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP) II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling

CNIH-2 facilitates ER export of AMPARs.

<p><b>A</b> Representative confocal images of OK cells stably expressing CNIH-2. Co-expression of dominant-negative Sar1 H79G prevents ER export of CNIH-2 leading to its redistribution into the ER. <b>B</b> Quantification of GluA1_o surface expression levels by extracellular epitope tagging in the presence of CNIH-2 and either wildtype (WT) Sar1 (white bar) or mutant Sar1 H79G (grey bar). Data are mean increases in surface expression levels by CNIH-2 ± SEM normalized to GluA1_o+Sar1 WT or GluA1_o+Sar1 H79G without CNIH-2, respectively. Asterisk marks a significant increase in surface expression of GluA1_o by co-expression of CNIH-2 (p<0.001, unpaired Student's t-test; n = 12 for both experimental groups). <b>C</b> Quantification of GluA1_o surface expression levels in the presence of CNIH-2 and either wildtype dynamin-1 (white bar) or dominant-negative dynamin-1 K44A (grey bar) inhibiting clathrin-dependent endocytosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030681#pone.0030681-Damke1" target="_blank">[38]</a>. Data are mean increases in surface expression levels by CNIH-2 ± SEM normalized as in B (n = 6 for both experimental groups).</p

CNIH-2 is rendered a surface membrane protein by assembly with AMPARs.

<p><b>A</b> Total (T), surface (S) and internal (I) populations of CNIH-2 in HeLa cells expressing either CNIH-2 alone (CTRL) or together with GluA1_o or GluA2_i, respectively. S is concentrated 10fold. Note that in the absence of GluAs, CNIH-2 cannot be detected in the surface fraction. However, it is robustly observed in the plasma membrane when co-expressed with GluAs (n = 4). <b>B</b> Total (T), surface (S) and internal (I) populations of CNIH-2 in dissociated hippocampal neurons (DIV 17) transduced with CNIH-2 (+) or GFP (−). S is concentrated 10fold. Both endogenous (−) and over-expressed (+) CNIH-2 can be detected on the cell surface (n = 5). <b>C</b> (Top) Representative current traces recorded in somatic outside-out patches excised from dissociated hippocampal neurons (DIV 16–21) over-expressing either GFP (CTRL, black) or CNIH-2 (CNIH-2, red) upon 1 ms (left panel) and 100 ms applications (right panel) of 10 mM glutamate. (Bottom) Quantification of deactivation and desensitization kinetics. Data are given as mean ± SD. Asterisk denotes a significant difference from control (p<0.01, unpaired Student's t-test; deactivation: n = 10 and 8 for CTRL and CNIH-2, respectively; desensitization: n = 19 and 8 for CTRL and CNIH-2, respectively).</p