Regulation of NMDAR function by tyrosine phosphorylation: The role of extracellular zinc and identification of the tyrosine phosphatase step as an endogenous regulator of NMDAR activity.

摘要

The NMDA subtype of glutamate receptor is a principal mediator of excitatory synaptic transmission within the CNS. Proper NMDA receptor (NMDAR) function is crucial for normal CNS processes, while dysfunction of NMDARs can lead to a host of nervous system disorders. NMDAR function is dynamically tuned to the state of the neuron by intracellular biochemical processes, including tyrosine phosphorylation/dephosphorylation. The tyrosine kinase Src physically associates with the NMDAR complex and enhances NMDAR activity. The work outlined herein addresses two aspects of NMDAR modulation by tyrosine phosphorylation: (1) the nature of the mechanism by which Src modulates NMDARs, and (2) the identity of the phosphotyrosine phosphatase (PTP) that counteracts Src regulation of NMDARs.;With recombinant receptors comprised of NR1-1a/NR2A orNR1-1a/2B subunits Src reduces voltage-independent Zn2+ inhibition. Thereby the function of recombinant NMDA receptors is potentiated by Src only when the Zn2+ levels cause tonic inhibition. In the first study of this thesis we investigated whether Src-induced potentiation of NMDARs in neurons proceeds via relief of Zn2+ inhibition. Whereas chelating extracellular Zn2+ blocked Src-induced potentiation of NR1-1a/2A receptors, Zn2+ chelation did not affect potentiation of NMDAR currents by Src applied into hippocampal neurons. Moreover, Src did not alter the Zn 2+ concentration-inhibition relationship for NMDAR currents. In dorsal horn neurons, chelating extracellular Zn2+ failed to prevent regulation of NMDA single-channel activity by endogenous Src and did not affect Src-mediated regulation of synaptic NMDARs. Thus Src potentiation of NMDAR currents is not mediated by reducing Zn2+ inhibition in hippocampal and dorsal horn neurons.;In the second study presented in this thesis we show that striatal enriched tyrosine phosphatase (STEP) is a component of the NMDAR complex. Functionally, exogenous STEP depressed NMDAR single-channel activity. STEP also depressed NMDAR-mediated synaptic currents whereas inhibiting endogenous STEP enhanced these currents. In hippocampal slices administering STEP into CA1 neurons did not affect basal glutamatergic transmission but prevented LTP induction. Conversely, inhibiting STEP enhanced transmission and occluded LTP induction through an NMDAR-, Src-, and Ca2+-dependent mechanism. Thus, STEP opposes Src to regulate NMDAR activity thereby acting as a tonic brake on synaptic transmission.