The list of methods used by the author to achieve the above objectives. More detailed descriptions of the methods can be found in the cited articles.
− preparation and culture of human embryonic kidney 293 cells (HEK293) and embryonic hippocampal neurons (Hemelikova et al., 2019; Kolcheva et al., 2021)
− transfection of cultivated HEK293 cells by the Magnet Assisted Transfection (MATra) technique (Hemelikova et al., 2019; Kolcheva et al., 2021)
− electrophysiological measurements using the patch-clamp technique in the whole- cell configuration (Hemelikova et al., 2019; Kolcheva et al., 2021)
− site-directed and/or multi-site-directed mutagenesis (Hemelikova et al., 2019)
− NMDA-induced excitotoxicity (Kolcheva et al., 2021)
− immunofluorescence microscopy – surface and total staining, image acquisition, and image processing (Kolcheva et al., 2021)
36 5. Results
- Hemelikova et. al, 2019
1) Using patch-clamp electrophysiology methods with rapid solution exchange we showed that disruption of the N440 and N674 N-glycosylation sites in the GluN1 subunit, as well as the N786 site in the GluN3B subunit, increasing the ratio of the peak tail current amplitude to the steady-state current. However, disruption of specific N-glycosylation sites within the GluN1 and GluN3A subunits did not affect functional properties of GluN1/GluN3A receptors.
2) Our electrophysiological results showed that variety of lectins, including ConA, WGA, and AAL, reduced GluN1 subunit-mediated desensitization. The strongest effect was observed with AAL and we showed that it is partly mediated by a single N-glycosylation site on each of the GluN3 subunits – N565 on GluN3A and N465 on GluN3B. However, no effect of lectins was observed when GluN1/GluN3 receptors were activated before the lectin treatment, suggesting that the modulatory effect of lectins might depend on the conformational state of the receptor.
In Hemelikova et. al, 2019 the author of this dissertation performed: i) electrophysiological experiments with GluN1/GluN3B receptors; ii) generation of GluN3B constructs with mutated N-glycosylation sites.
- Kolcheva et. al, 2021
1) We found that GluN1-M641I/GluN2A, GluN1-Y647S/GluN2A and GluN1- Y647S/hGluN2B receptors had reduced surface expression,both in HEK293 cells and in hippocampal neurons. All other NMDAR combinations, specifically GluN1- M641I/GluN2B, GluN1-M641I/GluN3A, GluN1-A645S/GluN2A, GluN1- A645S/GluN2B, GluN1-A645S/hGluN3A and GluN1-Y647S/GluN3A, were delivered to the cell surface at the same level as their corresponding WT.
2) Using whole-cell patch-clamp recordings, we calculated the EC50 values for glutamate and glycine in cells expressing WT and mutated GluN1/GluN2 receptors, as well as for glycine in cells expressing WT and mutated GluN1/GluN3A receptors.
GluN1-Y647S co-expressed with GluN2 or GluN3A subunits produced no measurable current. Our analysis showed that GluN1-M641I and GluN1-A645S co-
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expressed with GluN2 subunits does not affect the EC50 values for glutamate and glycine. The EC50 value for glycine of the GluN1-A645S/GluN3A receptor was similar to WT GluN1/GluN3A, but significantly increased for GluN1- M641I/GluN3A receptors. Moreover, GluN1-A645S/GluN3A receptors had a slower time constant of desensitization compared to WT GluN1/GluN3A.
3) We found no differences in Mg2+ sensitivity in cells expressing WT and mutated GluN1-M641I/GluN2A and GluN1-A645S/GluN2A receptors. Next, the IC50 value for memantine was significantly decreased for GluN1-M641I/GluN2A receptors and significantly increased for GluN1-A645S/GluN2A receptors in the presence of 1 mM Mg2+. Similar results were obtained from cultured hippocampal neurons expressing either WT or mutated GluN1 subunits.
4) Next, we examined how selected pathogenic mutations affect memantine SSI and the time constant of the recovery from SSI (τrecovery). Briefly, to induce SSI, cells were pretreated with 100 μM memantine before application of 1 mM glutamate. The current induced by glutamate was then compared with the currents induced without pretreatment with memantine. To quantify these data, we calculated the minimum ratio between SSI and control currents ("minimum ISSI/Icontrol"). Our analysis revealed that minimum ISSI/Icontrol and τrecovery for GluN1-4a-M641I/GluN2A receptors was similar to WT GluN1/GluN2A receptors. In contrast, we found that the minimum ISSI/Icontrol for GluN1-A645S/GluN2A receptors was significantly increased in comparison to WT GluN1/GluN2A receptors. We could not calculate τrecovery for GluN1-A645S/GluN2A receptors because of the relatively weak memantine-induced SSI.
5) We found that memantine differentially affected NMDA-induced excitotoxicity in neurons expressing mutated GluN1 subunits. Specifically, memantine significantly excitotoxicity in neurons expressing the YFP-GluN1-M641I subunit but was significantly less neuroprotective in neurons expressing the YFP-GluN1-A645S subunit in comparison to neurons expressing WT YFP-GluN1 subunit. These data indicate that specific pathogenic mutations in the M3 domain of the GluN1 subunit differentially affect the pharmacological sensitivity of NMDARs in hippocampal neurons.
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In Kolcheva et al., 2021 the author of this dissertation performed: i) immunofluorescence microscopy of surface NMDARs on the hippocampal neurons;
ii) all whole-cell patch-clamp recordings; iii) NMDA-induced excitotoxicity experiments.
- Skrenkova et al., 2019
1) We found that structural changes in glycine-binding sites, such as disruption of the S1 segment of GluN1 and GluN3A subunits, correlate with changes in the surface expression of NMDARs.
2) The presence of a clinically relevant D845N-GluN3A mutation leads to a dramatic reduction in the surface expression of NMDARs, both in heterologous COS-7 cells as well as rat hippocampal neurons.
In Skrenkova et al., 2019 the author of this dissertation performed whole-cell patch- clamp recordings from HEK293 cells transfected with the wild-type or mutated GluN1/GluN3A receptors.
- Skrenkova et al., 2020
1) Using whole-cell patch-clamp recording, we found that the pathogenic missense GluN1-S688Y mutation significantly increases the EC50 values for glycine and D- serine in GluN1/GluN2 receptors and significantly decreases desensitization of GluN1/GluN3A receptors.
2) Using immunofluorescence microscopy techniques, we showed that the pathogenic missense GluN1-S688Y mutation reduces surface expression of GluN3A-, but not GluN2B-containing NMDARs in cultured rat hippocampal neurons.
3) We observed that the GluN1-S688Y mutation decreases NMDA-induced excitotoxicity in cultured rat hippocampal neurons.
In Skrenkova et al., 2020 the author of this dissertation performed whole-cell patch- clamp recordings from primary hippocampal neurons expressing YFP-hGluN1-1a or YFP-hGluN1-1a-S688Y.
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