Regulation of NMDA receptor gating and trafficking by PSD-95

Date

2003

Authors

Lin, Ying

Journal Title

Journal ISSN

Volume Title

Publisher

ProQuest Dissertations & Theses

YU Faculty Profile

Abstract

NMDA (N-methyl-D-aspartate) receptors (NMDARs) are glutamate-gated ion channels that mediate synaptic transmission at excitatory synapses of the brain. Synaptic NMDARs are localized to p&barbelow;osts&barbelow;ynaptic d&barbelow;ensities (PSDs). The overall objective of this thesis research is to elucidate the molecular mechanisms by which PSD-95 regulates NMDAR gating and trafficking and modulation by protein kinases. To achieve this purpose, experiments involving patch-clamp and whole-cell recording, biotinylation western-blot analysis of cell surface proteins, immmunofluorescence and site-directed mutagenesis were carried out on Xenopus oocytes and HEK-293 cells expressing recombinant NR1/NR2A NMDARs. We chose NR1/NR2A receptors because they are the predominant NMDAR subtype expressed at mature excitatory synapses. Recombinant receptor systems provide expression of a homogenous population of NMDARs and geometric simplicity.;The first part of my thesis research focuses on regulation of NMDAR gating and trafficking by PSD-95. Single-channel recording from outside-out patches excised from Xenopus oocytes expressing NR1/NR2A receptors in the presence and absence of PSD-95 show that PSD-95 increases NMDA channel npo (the number of functional channel times open probability), with little or no change in single channel conductance, reversal potential or mean open time. I demonstrate that PSD-95 increases the number of functional NMDA channels. Moreover, PSD-95 increases the rate of decay of the NMDA-elicited current in the presence of MK-801, indicating an increase in the opening rate of NMDA channels. Removal of the PDZ binding motif in the NR2A subunit abolished the effects of PSD-95 on NMDA-elicited current, NMDAR surface expression and receptor internalization. These data suggest that physical interaction of NMDARs with PSD-95 is critical for PSD-95 regulation of NMDAR function and trafficking.;The second part of my thesis research investigates the molecular mechanism whereby PSD-95 reduces PKC potentiation of NMDA receptors. Truncation experiments indicate that the C-terminal domain of the NR2, rather than the NR1 subunit is responsible for PSD-95 actions. PSD-95 blocks PKC potentiation by occlusion of PKC-induced delivery of new NMDA channels to the plasma membrane and increase of NMDA channel opening rate. These mechanisms may be relevant to synaptic plasticity. (Abstract shortened by UMI.).

Description

Keywords

Neurosciences.

Citation

Source: Dissertation Abstracts International, Volume: 64-08, Section: B, page: 3691.;Advisors: R. Suzanne Zukin.