Regulation of NMDA receptor gating and trafficking

Abstract

The overall objective of this thesis research was to elucidate the molecular mechanisms underling the potentiation of NMDARs by activation of protein kinase (PKC) and by the G-protein coupled metabotropic glutamate receptor 1alpha (mGluR1alpha) and the action of spermine on NMDAR function. To achieve this purpose, experiments involving patch-clamp and whole-cell recording, Western analysis of cell surface proteins, and immunofluorescence were carried out on Xenopus oocytes expressing recombinant NMDARs and on cultured hippocampal neurons.;To elucidate the molecular mechanisms underlying potentiation of NMDARs by PKC, we recorded single channel activity in outside-out patches excised from oocytes expressing NR1--4b/NR2A receptors before and after treatment with the PKC-activating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). This isoform was chosen because NR1--4a and NR1--4b have the shortest C-terminal tail, and NR1--4b/NR2A and B have the highest cell surface expression and highest degree of PKC potentiation. To analyze independently the effects of PKC on number of functional channels in the membrane, N, and channel open probability, po, NMDA whole-cell currents were recorded during block by the quasi-irreversible open-channel blocker MK-801. PKC activation increased the number of channels per cell but did not significantly alter channel open probability. The increase of surface expression of NMDARs after PKC activation was confirmed by Western-blot analysis of biotinylated surface proteins and by immunofluorescence labeling with an antibody against an extracellular epitope of NMDARs in non-permeablized oocytes.;Activation of mGluRs modulates NMDAR activity under conditions of normal and enhanced synaptic activity. Electrophysiological and biochemical experiments were carried out on oocytes expressing recombinant receptors to elucidate the molecular mechanisms underlying mGluR1alpha-mediated potentiation of NMDARs. Single-channel recordings from excised outside-out patches showed an increase in the number of active channels times channel open probability (npo), with no change in mean open time, unitary conductance or reversal potential.;We further examined the action of spermine on recombinant NR1--4b/NR2B receptors. Single channel recordings showed that spermine reduced NMDA single-channel conductance in a concentration and voltage-dependent manner with partial relief of block evident at extreme membrane potentials. (Abstract shortened by UMI.).