STriatal Enriched Protein Tyrosine Phosphatase mediates Ethanol inhibition of NMDA Receptor Activity

We demonstrated previously that ethanol inhibition of NMDA receptor (NMDAR) function is accompanied by a reduction in tyrosine phosphorylation of Tyr1472 on the NR2B subunit, and this action of ethanol is attenuated by a broad spectrum tyrosine phosphatase inhibitor. Here we examined whether this ethanol inhibition of NMDAR activity was due to the actions of STriatal Enriched protein tyrosine Phosphatase (STEP) which has been shown to regulate NMDAR internalization by dephosphorylating Tyr1472 on the NR2B subunit. Using whole-cell recordings of pharmacologically isolated NMDAR-mediated excitatory post-synaptic currents (NMDA EPSCs) from hippocampal CA1 pyramidal neurons, we show that intracellular infusion of a substrate-trapping inactive form of STEP (TAT-STEP C/S) significantly blocks ethanol inhibition of NMDA EPSCs. Ethanol does not inhibit NMDA EPSCs or LTP in neurons from STEP knockout mice, but its effect is restored after acute intracellular delivery of wild type TAT-STEP, suggesting that STEP mediates ethanol inhibition of NMDAR function. The majority of excitatory synaptic transmission in the mammalian CNS is mediated by the neurotransmitter glutamate, which activates postsynaptic α-amino-3-hydroxy-5methyl-4-isoxalone propionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes of ionotropic glutamate receptors. NMDA receptors (NMDARs) in the hippocampus consist of NR1/NR2A, NR1/NR2B, and NR1/NR2A/NR2B receptor subunit complexes. While NR1 subunits are required to form an active ion channel, incorporation of the various NR2 subunits regulate NMDAR channel activity by altering the channel kinetics and/or mediating the differential effects of pharmacological agents including ethanol. Acute ethanol application inhibits NMDAR channel activity. In the hippocampus, this inhibitory effect of ethanol on NMDARs is widely thought to underlie both the acute amnestic effects of ethanol and also, in part, the addictive nature of ethanol. However, the precise molecular mechanisms underlying ethanol’s inhibition of NMDARs have not been well-understood. We previously demonstrated that ethanol inhibition of NMDAR function is associated with dephosphorylation of tyrosine residues on the NR2A and NR2B subunits. In particular, ethanol reduced phosphorylation of a site, Tyr1472, on the NR2B subunit which regulates endocytosis of NMDARs. Moreover, ethanol-induced inhibition of NMDAR function was prevented by bath application of the protein tyrosine phosphatase (PTP) inhibitor, bpV(phen). Based on these and other findings that showed ethanol reduced the tyrosine phosphorylation of NR2 subunits in the cortex, we proposed that ethanol inhibition of NMDAR activity is mediated by a PTP. PTPs are a large family of enzymes that are broadly divided into receptor-like PTPs and intracellular PTPs, and are implicated in a number of neuronal functions. STriatal Enriched protein tyrosine Phosphatase (STEP) is a brainspecific PTP expressed in the striatum, hippocampus and cortex among other brain regions. Within neurons, STEP is localized to the endoplasmic reticulum and in postsynaptic densities of glutamatergic synapses. Of the four STEP isoforms, STEP61 is the only one expressed in the hippocampus. STEP61 forms a complex with the NMDAR, reduces its activity, and opposes the induction of long-term potentiation (LTP), a form of plasticity widely thought to play a role in learning and memory. The current hypothesis for STEP function is that it blocks the development of synaptic strengthening. Consistent with these findings, enhanced STEP activity is associated with dephosphorylation of the Tyr1472 residue on the NR2B subunit, a site that is dephosphorylated by ethanol. In addition, NMDAR trafficking to synaptic membranes is increased after RNA interference of STEP. Based on these data, we predicted that STEP mediates the inhibitory effects of ethanol on NMDARs in various brain regions, including the hippocampus. We utilized whole-cell recordings of pharmacologically isolated NMDA EPSCs in both rat and mouse hippocampal slices. Moreover, we utilized the recently generated STEP KO mice to further test the hypothesis. Our results show that STEP is responsible, at least in part, for the inhibitory effects of ethanol on hippocampal NMDAR activity.