Regulation of NRI/NR2B NMDA receptor function by Thrombin

Abstract

N-methyl-D-aspartate (NMDA) receptor is a subfamily of the glutamate receptors in the central nervous system (CNS) that is involved in the mediation of many physiological activities such as learning and memory. However, overactivation of the NMDA receptors results in excitotoxicity that is often involved in the progression of neuronal cell death in diseases such as ischemic stroke. As such, NMDA receptors are tightly regulated by endogenous mediators. In particular, the serine protease, thrombin, which is observed in the astrocytes and neurons in the CNS, is involved in modulating the function of the NMDA receptors through the activation of the protease-activated receptor (PAR)-1. Direct interaction with the NMDA receptors by thrombin has yet been fully characterized and determined. The aim of the thesis is, thus, to investigate the possibility of direct interaction between thrombin and the NMDA receptors and the possible effects in the modulation of the NMDA receptors. In this study, thrombin was observed to interact with the NR2B of the NMDA receptors from rat brain lysate (RBL) and synaptic plasma membrane (SPM) preparations. Based on epitope mapping and the sizes of the fragments (30 kDa fragment and 150 kDa fragment) observed, thrombin was hypothesized to cleave NR2B at the amino terminal domain (ATD). To identify the site of interaction of NR2B with thrombin, the NR2B ATD was expressed as a soluble recombinant fusion protein (MBP-ATD2B) and was subjected to thrombin treatment. N-terminal sequencing of the thrombin-cleaved product deduced the cleavage site to be Lys318 at the NR2B ATD. The cleavage site was further confirmed through the absence of cleavage on the MBP-ATD2B(K318A). Thrombin cleavage studies performed on cortical neuronal culture also demonstrated that thrombin could cleave NR2B expressed in heteromeric NMDA receptors complex. Through two-electrode voltage clamp (TEVC) recordings on Xenopus laevis oocytes expressing NR1/NR2B receptors, it was also observed that a reducing environment, one of the conditions of ischemic stroke, resulted in more efficient thrombin cleavage of NR2B, as demonstrated by a reduction in ifenprodil inhibition. Molecular dynamics simulation based on the NR2B ATD crystal structure also provided an insight into how a reducing environment exposed the Lys318 to the extracellular milieu, allowing for interaction with thrombin. In the final part of the thesis, the various effects of the cleavage were investigated through TEVC recordings. In particular, the deletion construct, with the ATD region up to Lys318 removed (NR2B-¿ATD-K318) demonstrated an increase in the ifenprodil IC50 and a change in the EC50 of glycine and the efficacy of D-cycloserine when co-expressed with NR1. Interestingly, unlike ifenprodil, glycine and D-cycloserine are ligands binding to the NR1 ligand binding domain (LBD) but not the NR2B ATD. These results suggested allosteric modulation of the ATD of NR2 on LBD of NR1 and the importance of the ATD in modulating receptor function. Taken together, this study had discovered thrombin cleaved NR2B at a specific site at the ATD, which could lead to the alteration of NMDA receptor function. This study had provided an insight on the possible modulation of NMDA receptors through interaction with proteases, in particular, thrombin.