Kainate receptors (KARs) serve a crucial role as modulators of synaptic transmission and plasticity and their dysfunction has been linked to several disease states such as epilepsy, chronic pain and neurodegenerative diseases. Trafficking of KARs is modulated in a number of ways, including ligand binding and subunit composition, with GluKl-3 trafficking as homomeric receptors and GluK4/S only trafficking as heteromeric receptors following assembly with GluKl-3. In this study we used molecular modelling of the GluKl-3 5152 domains to identify amino acid residues which are likely to be critical for ligand binding. Using site directed mutagenesis we have changed a threonine residue to valine in GluKl-3 (T70SV, T690V, T692V) and AS18L in GluK2. Residues were also identified that differed between the GluKl-3 subunits and could serve to determine ligand selectivity, and also served as control mutations as they have retained ligand binding. These include a reciprocal mutation at the Asn/5er residue (GluK1 N73S5/5736N, GluK2 5720N/N7215 and GluK3 N7225) and the Thr/Ala residue (GluK1 TS33A, GluK2 AS18T). Residues were also determined that control UBP161 GluK1 specificity this was identified to be possibly due to the 5674 residue, which in GluK3 the equivalent residue is A660, which when mutated to Ser increased UBP161 affinity for GluK3 consistent with a subunit switch. Another ligand UBP32S, a biotinylated prototype was tested and found to selectively isolate GluK1 receptors. The predicted changes in ligand binding activities were confirmed using radioligand binding assays. In line with previous studies of iGluRs, homomeric KARs with impaired ligand binding sites were retained in the ER, in both biotinylation and endoglycosidase analysis and showed no detectable Ca2+ currents. Assessment of the stability and expression levels of these KAR assemblies by inhibition of protein synthesis, revealed that although loss of . ligand binding decreased expression levels, it did not significantly increase the degradation rate of the KAR assemblies with impaired ligand binding. However, impaired subunits were able to co-assemble with GluKS, overruling the ER retention of both GluK2 with impaired ligand binding, and GluKS. Determination of the critical residues that determine surface targeting and ligand selectivity between the GluKl-3 subunits is crucial. As it allows the design of specific ligands, that can be targeted to these specific residue differences, leading to the production of more subunit specific ligands. In addition the study of the GluK2 and 5 heteromeric assemblies, allows the functional effects of ligands on the GluKS subunits to be analysed when the GluK2 subunit has no functional responses due to a lack of ligand binding.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:559234 |
| Date | January 2012 |
| Creators | Scholefield, Caroline |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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