Synaptic zinc inhibition of NMDA receptors depends on the association of GluN2A with the zinc transporter ZnT1 – Science Advances

INTRODUCTION

Zinc is a neuromodulator that regulates glutamatergic, -aminobutyric acidmediated (GABAergic), and glycinergic synaptic transmission (17); short- and long-term synaptic plasticity (2, 812); auditory processing; and acuity for sensory stimulus discrimination (1315). The zinc transporter ZnT3 (Slc30a3) packages zinc into presynaptic vesicles of large populations of excitatory neurons in many brain regions, including the cerebral cortex, hippocampus, amygdala, and dorsal cochlear nucleus (DCN) (16). During synaptic activity, vesicular zinc is released from ZnT3-containing terminals (1, 17) and diffuses across the synaptic cleft (4) to modulate a number of postsynaptic receptors (3, 6, 18, 19), including the highly zinc-sensitive N-methyl-d-aspartate receptor (NMDAR) (2, 4, 20).

GluN2A-containing NMDARs are the major postsynaptic targets of synaptically released zinc due to their sensitivity to nanomolar concentrations of extracellular zinc, which inhibit receptor function (20). It is generally accepted that zinc release alone provides sufficient accumulation of the metal in the synaptic cleft to account for the observed zinc inhibition of GluN2A-containing NMDARs (2, 4, 9). However, this model only considers ZnT3s contribution to activity-dependent vesicular zinc inhibition of NMDARs when there are 24 known unique zinc transporters (10 Slc30a and 14 Slc39a) (21) that may be involved in zincs spatial distribution in synapses. Little is known, however, how zinc transporters other than ZnT3 influence synaptic zincs actions upon its receptor targets.

ZnT1 (Slc30a1), a cell membrane transporter that shuttles zinc from the cytoplasm to the extracellular space, not only localizes to the postsynaptic density (22) but also binds directly to the GluN2A subunit of NMDARs (23). This positions ZnT1, in concert with the presynaptic ZnT3 transporter, as a likely co-regulator of synaptic zinc concentration and function in the synaptic cleft. Here, we developed a cell-permeant peptide to disrupt ZnT1-GluN2A binding and used ZnT3 null mice and zinc chelation to assess the contribution of ZnT1 into synaptic actions of zinc. Our studies reveal a previously unknown interplay between ZnT3- and ZnT1-dependent zinc transport to inhibit NMDAR-mediated neurotransmission.

To study the effects of ZnT1 on zinc inhibition of NMDARs, we designed a cell-permeant peptide aimed at disrupting the ZnT1-GluN2A interaction. First, we constructed a peptide spot array spanning 74 amino acids of the C-terminal domain (residues 1390 to 1464) of mouse GluN2A (UniProt no.: P35436), previously shown to be necessary for ZnT1-GluN2A binding (23). The array consisted of sixty-one 15-mers, each sequentially overlapping by 14 amino acids, similar to procedures described earlier by our groups for other protein-protein interaction systems (24, 25). Next, we probed the peptide spot arrays with Flag-tagged ZnT1-enriched human embryonic kidney (HEK) cell lysates and then visualized and quantified ZnT1 binding with immunofluorescence against the Flag tag (Fig. 1A). This approach identified three regions of ZnT1 binding, spanning peptide numbers 2 to 8, 40 to 42, and 48 to 52 in Fig. 1 (A and B). We focused on the broadest binding peak (peptides 2 to 8; Fig. 1B, in red), which included a common nineamino acid sequence among the peptides with high ZnT1 binding (NDSYLRSSL, corresponding to GluN2A residues 1397 to 1406). This nineamino acid sequence is conserved in mouse, rat, and human GluN2A sequences (isoform 1; UniProt no.: rat, Q00959; human, Q12879). Last, this peptide and a scrambled control (SNLSDSYLR; Fig. 1B, inset) were conjugated to the trans-activator of transcription (TAT) cell-penetrating peptide (YGRKKRRQRRR) to endow them with membrane permeability. As it was designed to prevent ZnT1-GluN2A binding, the peptide and its scrambled control will here be referred to as N2AZ and scN2AZ, respectively.

(A) Peptide spot array was composed of sixty-one 15-mers spanning the GluN2A C-terminal region (residues 1390 to 1464) with 14amino acid overlapping sequential sequences used to identify regions of high ZnT1 binding. A representative array is shown, with corresponding peptide numbers denoted below the blot. Sequences for each peptide number are shown in (B). The peptides denoting the broadest ZnT1-binding region are outlined in red. (B) Mean SEM (n = 4) of ZnT1-binding intensity for each GluN2A-derived peptide. Inset: Peptide sequences flanking a region of high ZnT1 binding (peptide numbers 2 to 8, in red) were used to determine the shared peptide sequence of the ZnT1-binding peptides. Sequence in light gray represents the cell-permeable HIV TAT domain sequence. The red sequence represents the final peptide, and the blue sequence represents its scrambled control. Both peptides were conjugated to TAT to create our experimental (N2AZ) and control (scN2AZ) peptides.

To confirm that N2AZ prevents GluN2A-derived sequences from binding to ZnT1, the peptide spot assay was repeated in the presence of either N2AZ or scN2AZ (100 M). We noted that N2AZ reduced ZnT1 binding to the spot array when compared to scN2AZ control (fig. S1). These results indicate that N2AZ can disrupt the ZnT1-GluN2A association.

In the next set of experiments, we used rat cortical cultures to determine whether N2AZ treatment was sufficient to disrupt ZnT1-GluN2A association in neurons. We first verified that ZnT1 mRNA was expressed in cortical cultures using quantitative polymerase chain reaction (PCR). ZnT1 mRNA expression increased over the first 4 weeks in culture (fig. S2), with maximal expression levels observed after 19 days in vitro (DIV). The developmental expression profile of ZnT1 parallels, to some extent, that of GluN2A expression in the same preparation, with this NMDA receptor subunits mRNA becoming highly detectable after 15 DIV (26). To quantify ZnT1-GluN2A interactions in the cultures, we used a proximity ligation assay (PLA), a method that produces discrete fluorescent puncta when target proteins are within 40 nm of one another, thus revealing putative protein-protein interactions (27). Cultures (20 to 27 DIV) were treated overnight with either scN2AZ or N2AZ (3 M) before performing PLA. To visualize neurons, cultures were also immunostained for neuron-specific microtubule-associated protein 2 (MAP2). We observed that PLA puncta localized along neuronal dendrites, consistent with previous findings localizing ZnT1 to postsynaptic density (Fig. 2A) (22, 23). We found that N2AZ treatment reduced the number of PLA puncta when compared to sister cultures treated with scN2AZ (paired t test, P = 0.004; n = 4; Fig. 2B). These results indicate that N2AZ effectively disrupts ZnT1-GluN2A interactions in cultured neurons.

(A) Representative images of rat cortical cultures following PLA between GluN2A and ZnT1. The PLA immunofluorescently labeled sites of interaction between GluN2A and ZnT1 (white puncta). In addition, MAP2 is immunofluorescently labeled in red to visualize neuron morphology. Scale bar, 50 m. Top row shows PLA following overnight exposure to 3 M scN2AZ, while bottom row shows PLA following 3 M N2AZ treatment. Insets show the localization of PLA puncta along a MAP2-stained dendrite. (B) Quantification of PLA puncta per 100 m2 in sister cortical cultures treated overnight with 3 M N2AZ or scN2AZ shows that N2AZ significantly reduced the number of ZnT1-GluN2A interactions compared to scN2AZ (paired t test, P = 0.0044; n = 4). Gray-filled circles indicate the quantification of representative images in (A). Error bars indicate mean SEM.

Zinc is an allosteric inhibitor of NMDARs through high-affinity binding on the extracellular, N-terminal domain of the GluN2A subunit (4, 20). As ZnT1 shuttles neuronal intracellular zinc to the extracellular space (28), we hypothesized that ZnT1 functionally localizes zinc in close proximity to its GluN2A-binding site and thereby contributes to the inhibition of NMDARs by the metal. To test this hypothesis, we treated DIV 20 to 27 cortical cultures overnight with either N2AZ or scN2AZ (3 M) before recording NMDAR-mediated currents. These currents were evoked by photolytic uncaging of 4-methoxy-7-nitroindolinyl (MNI)caged glutamate (40 M) along the dendrites of neurons (Fig. 3A). Neurons were held at 70 mV in the absence of extracellular Mg2+ to prevent block of NMDARs by this cation and in the presence of DNQX (20 M) to block AMPA receptor (AMPAR) currents. Zinc inhibition was determined by measuring the extent of NMDAR excitatory postsynaptic current (EPSC) potentiation following application of ZX1 (3 M), a fast, high-affinity, zinc-specific, extracellular chelator (3, 4, 9). We observed that extracellular zinc chelation with ZX1 produced a potentiation of NMDAR-mediated currents in scN2AZ-treated neurons (37.40 11.63% potentiation; n = 10; P = 0.02, paired t test of peak responses before and after ZX1), likely reflective of a background tonic zinc inhibition (Fig. 3, B and C). In contrast, N2AZ-treated neurons did not show any ZX1 potentiation of NMDAR-mediated currents (1.34 3.48%, n = 9, P = 0.62, paired t test; scN2AZ versus N2AZ, P = 0.01, unpaired t test; Fig. 3, B and C ). Together, these results indicate that ZnT1-GluN2A binding is critical for endogenous zinc inhibition of NMDAR-mediated currents in cortical neuronal cultures.

(A) Representative image of a neuron in cortical culture filled with Alexa 548 during whole-cell recording. Blue asterisk represents one example location of laser photolysis of MNI-caged glutamate (40 M, 1-ms pulse) used to evoke EPSCs. (B) Sample traces of NMDAR currents, averaged over four sweeps, evoked by photolysis of MNI-caged glutamate in cultured cortical neurons held at 70 mV in Mg2+-free solution, before (blue, scN2AZ; red, N2AZ; 3 M, treated overnight) and after application of ZX1 (black; 100 M). (C) ZX1 potentiation of NMDAR currents was significantly diminished in N2AZ-treated cells versus scN2AZ control (unpaired t test, P = 0.01; n = 10, 9). Bar graphs represent the average potentiation of responses 5 min after ZX1 application. Error bars indicate mean SEM. *P < 0.05.

To investigate whether ZnT1 contributes to synaptic zinc inhibition of NMDARs, we performed electrophysiological recordings in acutely prepared brain slices of the DCN, an auditory brainstem nucleus containing parallel fibers with zinc-rich synaptic terminals (29). In response to parallel fiber stimulation, synaptic zinc inhibits both NMDA and AMPA-mediated synaptic currents in cartwheel cells (3, 4), interneurons in the molecular layer of the DCN. In these synapses, at 20-Hz synaptic stimulation frequency, zinc chelation with ZX1 does not potentiate NMDARs in ZnT3 null [knockout (KO)] mice, supporting the idea that ZnT3-dependent, extracellular, synaptically released zinc is necessary for zinc inhibition of NMDARs (4).

Here, we stimulated parallel fibers at 20 Hz and isolated NMDAR EPSCs by voltage-clamping cartwheel cells at +40 mV to relieve the Mg2+ block while recording in the presence of DNQX (20 M). Slices were incubated with either scN2AZ or N2AZ (3 M) for at least 1 hour before ZX1 application (100 M). We found that N2AZ significantly reduced the ZX1 potentiation of NMDAR EPSCs when compared to scN2AZ (N2AZ: 19.39 5.82%, n = 14 versus scN2AZ: 47.30 10.14%, n = 9; unpaired t test, P = 0.02; Fig. 4, A to C). This result indicates that, contrary to the decades-long current model, synaptically released zinc alone is necessary but not sufficient to account for complete inhibition of NMDAR by the metal. Instead, these results show that synaptic zinc inhibition of NMDARs also requires the association of ZnT1 with GluN2A.

(A and D) Sample traces of NMDAR EPSCs, averaged over five sweeps, evoked in cartwheel cells in response to five pulses at 20-Hz (A) or 100-Hz (D) stimulation frequency of parallel fibers. Before (blue, scN2AZ; red, N2AZ; 3 M, treated 1 hour before recording) and after application of ZX1 (black; 100 M). (B and E) Time course of NMDAR EPSCs, normalized to a 5-min baseline before addition of ZX1. Dotted line marks 100% of baseline. (C and F) Group data show that ZX1 potentiation of EPSCs was significantly reduced in N2AZ- versus scN2AZ-treated slices for 20-Hz stimulation (unpaired t test, P = 0.02; n = 14, 9) and 100-Hz stimulation (unpaired t test, P = 0.02; n = 14, 9). Bar graphs represent the average potentiation of responses 10 to 15 min after ZX1 application. Error bars indicate mean SEM. *P < 0.05.

Knowing that synaptic zinc inhibition of NMDARs is abolished in ZnT3 KO mice (4), we next tested the actions of N2AZ in wild-type (WT) and ZnT3 KO mice. We found that ZX1 had no significant effects on NMDAR EPSCs in either KO or WT N2AZ-treated slices (KO: 11.9 6.90% potentiation, n = 6, one-sample t test, P = 0.13; WT: 5.60 4.38% potentiation, n = 8, one-sample t test, P = 0.27; fig. S3). This finding indicates that in the absence of the association of ZnT1 with GluN2A, the presence of vesicular zinc is of no consequence to zinc inhibition of NMDARs in either WT or ZnT3 KO mice, further supporting our main hypothesis on the crucial role of GluN2A-ZnT1 association on zinc inhibition of NMDARs.

We previously reported that at high-frequency trains of synaptic stimulation (100 to 150 Hz), ZX1 potentiates NMDAR EPSCs in WT mice to the same extent as in ZnT3 KO mice, indicating that an additional, ZnT3-independent source of zinc, referred to as tonic (4), also inhibits NMDAR EPSCs. As such, we next evaluated the contribution of the ZnT1-GluN2A interaction in response to high-frequency stimulation synaptic stimulation. At 100-Hz stimulation frequency, we found that N2AZ treatment eliminated ZX1 potentiation of NMDAR EPSCs, suggesting that ZnT1-GluN2A interaction is also required for tonic, ZnT3-independent zinc inhibition (1.95 4.33%; n = 14; P = 0.45, paired t test of responses before and after ZX1; Fig. 4, D to F ). Together, our results indicate that ZnT1-GluN2A binding is critical for both ZnT3-dependent and ZnT3-independent inhibition of NMDARs.

In addition to blocking NMDAR EPSCs, synaptically released zinc inhibits AMPAR EPSCs in cartwheel cells (3). To test whether the aforementioned actions of N2AZ are specific for NMDAR EPSCs, we measured the effect of N2AZ and scN2AZ on zinc inhibition of AMPAR EPSCs. We observed that ZX1 potentiated AMPAR EPSCs to a similar extent regardless of the treatment with either scN2AZ or N2AZ (scN2AZ: 30.3 10.24%, n = 6; N2AZ: 29.3 6.70, n = 6; unpaired t test, P = 0.93; Fig. 5, A and B), indicating that N2AZ reduces zinc inhibition of NMDAR EPSCs without affecting AMPAR EPSCs.

(A) Cartwheel cells AMPAR EPSCs (five sweep averages), following parallel fiber stimulation (blue, scN2AZ; red, N2AZ; 3 M throughout this figure) in the absence and presence of ZX1 (black; 100 M). (B) ZX1 potentiation of AMPAR EPSCs (n = 3) is not different between treatments. (C) Sample traces of AMPAR EPSCs (50-ms interstimulus interval). (D and E) Average paired-pulse ratio (D) and coefficient of variance (CV; E) are not different between treatments (mean SEM; n = 3). (F) FluoZin-3 fluorescence in HEK293 cells transfected with vector (black), ZnT1 with scN2AZ treatment (blue), or ZnT1 with N2AZ treatment (red). Zinc (1 M, with 5 M pyrithione) treatment increases intracellular zinc (black bar). Zinc efflux was measured (box) as decreases in fluorescence. (G) Average showing the change in fluorescence after zinc washout. (H) Zinc efflux rates as the slope of the average fluorescence traces in (G). ZnT1-transfected cells exhibited greater zinc efflux compared to vector-transfected controls (one-way ANOVA/Tukey N2AZ versus vector, scN2AZ versus vector; P < 0.0001, n = 4 to 5); scN2AZ and N2AZ treatments do not differ. (I) Glutamate (1 mM, black bar)evoked currents in GluN1/2A-transfected HEK-tsa201 cells (60 mV; Mg2+-free) with increasing concentrations of zinc (1 to 300 nM). (J) Zinc inhibition curves; insets = IC50s (mean SEM; n = 5), vehicle-treated (black), scN2AZ-treated (3 blue), and N2AZ-treated (red) cells; all are similar (P = 0.5).

We also evaluated whether a change in presynaptic release of glutamate contributes to the observed actions of N2AZ on NMDAR-mediated synaptic currents. We used two independent measures of release probability: paired-pulse ratio (PPR) and the coefficient of variance (CV). Both measures vary inversely with probability of release. For PPR measurements, we applied two stimuli in rapid succession (50-ms interstimulus interval) to compute the amplitude ratio of the second to the first EPSC. We calculated CV as the SD of a series of EPSCs divided by their mean amplitude. We found that scN2AZ and N2AZ altered neither PPR nor CV (PPR: scN2AZ: 2.18 0.26, n = 3; N2AZ: 2.29 0.07, n = 5, unpaired t test, P = 0.59; CV: scN2AZ: 0.28 0.030, n = 3; N2AZ: 0.26 0.031, n = 5, unpaired t test, P = 0.76; Fig. 5, C to E). As such, N2AZs effects on zinc inhibition of NMDAR-mediated synaptic currents are not associated with changes in presynaptic glutamate release probability. These observations also rule out potential actions of N2AZ either on zinc signaling via the metabotropic zinc receptor GPR39 or on NMDAR-dependent, metabotropic activation of pannexin-1, as both signaling mechanisms are associated with changes in presynaptic glutamate release probability (11, 30).

To control for potential effects of N2AZ on the ZnT1 transporter activity, we next examined whether N2AZ modifies transport itself. Following intracellular zinc loading, we measured decreases in intracellular zinc levels over time as a readout of zinc transport in HEK293 cells previously transfected with a plasmid encoding ZnT1 or with an empty vector. We used FluoZin-3 fluorescence to measure intracellular zinc levels. FluoZin-3loaded cells were briefly treated with zinc pyrithione (1 M Zn2+ and 5 M sodium pyrithione) to increase intracellular zinc concentrations until the fluorescent signal reached a maximum, steady-state level (Fig. 5F). Zinc efflux was then measured as the decrease in FluoZin-3 fluorescence (Fig. 5G). As expected, ZnT1-expressing cells showed significantly more zinc efflux than vector-transfected controls [one-way analysis of variance (ANOVA), P 0.0001; Tukey multiple comparisons, N2AZ versus vector, scN2AZ versus vector, P 0.0001; Fig. 5H]. However, the rate of zinc efflux was not different in ZnT1-expressing cells treated with either scN2AZ or N2AZ [0.0002273 (F/F0)/s, n = 5] or scN2AZ [0.0002327 (F/F0)/s, n = 4] (Tukey multiple comparisons, N2AZ versus scN2AZ, P = 0.97), indicating that N2AZs actions on zinc inhibition of NMDARs cannot be explained by alterations in ZnT1 zinc transport activity.

To control for potential effects of N2AZ on NMDAR affinity for zinc itself, we measured NMDAR inhibition by exogenous zinc application onto HEK cells previously transfected with plasmids encoding GluN1 and GluN2A. Exogenous, extracellular zinc was applied across a wide range of concentrations (1 to 300 M) using a multi-barreled rapid-perfusion system while recording glutamate (1 mM)evoked steady-state GluN1/2A receptor current. The calculated IC50s (median inhibitory concentrations) for zinc block in vehicle, scN2AZ-treated, or N2AZ-treated cells were not different across the three treatments, indicating that N2AZ does not affect zincs affinity for GluN1/2A receptors (IC50 in nM; vehicle: 21.8 2.1, n = 5; scN2AZ: 23.4 2.4, n = 5; N2AZ = 25.6 2.2, n = 5; ordinary one-way ANOVA, P = 0.4996). Taking all of these results together, we conclude that N2AZ reduces zinc inhibition of NMDARs by disrupting the ZnT1-GluN2A interaction, without affecting glutamate release, ZnT1-dependent zinc transport, or zinc affinity for GluN1/2A receptors. Moreover, these results suggest that N2AZ does not have either toxic or nonspecific effects.

The simplest model to explain our results thus far is that the ZnT1-GluN2A interaction is necessary for zinc inhibition of NMDARs by transporting zinc from the cytoplasm of the postsynaptic cell to the extracellular space and in close proximity to the NMDAR. This model predicts that postsynaptic intracellular zinc is therefore also necessary for zinc inhibition of NMDARs. Because ZX1 is cell impermeant, we opted to selectively chelate intracellular zinc by including ZX1 (100 M) in the recording pipette, allowing us to test the contribution of intracellular zinc to synaptic zinc inhibition of NMDARs. ZX1 in the recording pipette was allowed to diffuse into the patched cell for at least 30 min before applying extracellular ZX1. We observed that intracellular ZX1, applied via the recording pipette, blocked extracellular ZX1 potentiation of NMDAR EPSCs (5.23 4.22%; n = 6; P = 0.64, paired t test of responses before and after ZX1; Fig. 6, A and B). In contrast, control experiments, without ZX1 in the recording pipette and assessed 30 min before applying extracellular ZX1, showed robust potentiation of NMDAR EPSCs (Fig. 6, A and B: 32.10 6.36%; n = 6; P = 0.009, paired t test of responses before and after ZX1; Fig. 6C: intracellular ZX1 versus control; P = 0.006, unpaired t test). This result indicates that intracellular postsynaptic zinc is required for synaptic zinc inhibition of NMDARs. Together, these results show that this intracellularly originated zinc pool along with the ZnT3-dependent extracellular zinc are both necessary for zinc inhibition of NMDAR EPSCs. As current models have been suggesting that zinc inhibition of synaptic NMDARs depends exclusively on presynaptically released extracellular zinc, this is an important finding that changes this dogma and significantly advances our understanding of a fundamental synaptic mechanism of neuromodulation.

(A) Sample NMDAR EPSCs at +40 mV, average of five sweeps, in response to five pulses at 20-Hz stimulation frequency, before and after application of extracellular ZX1 (100 M) in control (no intracellular ZX1) and in 100 M intracellular ZX1. (B) Time course of NMDAR EPSCs normalized to a 5-min baseline in control (black) and intracellular ZX1 (green) showing the effect of ZX1 on NMDAR EPSCs. Dotted line marks 100% of baseline. (C) Group data show that intracellular ZX1 significantly reduced extracellular ZX1 potentiation of NMDAR EPSCs compared to control [unpaired t-test, P = 0.006, n = 6 (control), 8 (intracellular ZX1)]. Bar graphs represent the average potentiation of responses 15 to 20 min after ZX1 application. **P < 0.01. (D) Sample traces of NMDAR EPSCs at +40 mV, average of five sweeps, in response to five pulses at 20-Hz stimulation frequency, before and after application of extracellular ZX1 (100 M) in control (0.01% DMSO) and nimodipine (20 M nimodipine in 0.01% DMSO), 20 min before beginning of recordings. (E) Time course of NMDAR EPSCs normalized to 5-min baseline in control (black) and extracellular nimodipine (orange) before and after ZX1 application (black bar, above). Dotted line marks 100% of baseline. (F) Group data show that ZX1 potentiation was not significantly different between nimodipine and control [unpaired t test, P = 0.99; n = 6 (control), 4 (nimodipine)]. Bar graphs represent the average potentiation of responses 10 to 15 min after ZX1 application. Error bars indicate mean SEM.

We next examined whether the well-established routes of entry for zinc into neurons, including calcium-permeable AMPAR (31) and l-type calcium channels (32), mediate potential translocation of synaptic zinc into the cytoplasm of cartwheel cells. AMPARs were immediately ruled out by the fact that all our experiments were performed in the presence of DNQX. As previous reports have shown that l-type calcium channels can also interact with ZnT1 (33), we examined whether these channels contribute to synaptic zinc inhibition of NMDARs. We applied nimodipine (20 M) for at least 20 min before recordings to inhibit l-type calcium channels and measured zinc inhibition of NMDAR responses. We observed no significant differences in ZX1 potentiation of NMDAR EPSCs between nimodipine-treated slices (23.6 6.9%, n = 4; Fig. 6, D to F) and vehicle-treated [dimethyl sulfoxide (DMSO)] slices (23.6 9.4%; n = 6; unpaired t test, P = 0.997), indicating that the putative translocation of synaptic zinc to postsynaptic neurons via l-type calcium channels does not contribute to zinc inhibition of NMDARs.

Current models suggest that zinc inhibition of synaptic NMDARs depends exclusively on presynaptically released zinc. In contrast, our results indicate that zinc inhibition of NMDAR EPSCs also requires postsynaptic zinc and the presence of GluN2A-ZnT1 association. Our results demonstrate that the physical dissociation of GluN2A and ZnT1 by the newly developed peptide N2AZ diminished the inhibitory actions of synaptic zinc on NMDAR EPSCs. Moreover, chelation of postsynaptic intracellular zinc abolished zinc inhibition of NMDARs. Before the work presented here, it had been generally assumed that zinc cleft concentrations following its synaptic release were sufficient to directly inhibit NMDAR function (1, 2, 4, 9, 16), without involvement of the transport pathway uncovered by our work. However, here, we show that ZnT1-GluN2A association is also necessary for zinc to be rapidly localized to physiologically relevant microdomains in very close proximity to the GluN2A-containing NMDARs. This is highly reminiscent of calcium microdomains that have been postulated for a number of synaptic functions, including rapid synaptic release of neurotransmitters (34). Whether similar transport processes are in place for synaptic zinc to activate or modify other known postsynaptic targets for the metal, including the metabotropic zinc receptor GPR39 (19) or AMPAR-mediated synaptic currents (3), remains to be determined.

Why the DCN synapses need both extracellular and intracellular zinc for synaptic zinc inhibition of NMDARs? This may be the result of the complex nature of zinc as a signaling molecule itself (9, 35). As alluded to earlier, zinc is a promiscuous ligand that acts on a variety of postsynaptic targets (3, 6, 11, 18, 36). Moreover, not all vesicles at zinc-rich synaptic terminals contain zinc (37, 38), and zinc-containing vesicle release probability can change with varying levels of activity (37, 39). Therefore, maintaining adequate signaling requires precise spatial zinc regulation, in addition to presynaptic release. The ZnT1-GluN2A interaction may be reflective of a system that harnesses and directs zincs signaling properties while supplying and maintaining specificity of action for a given activity level. As NMDAR function is regulated by subunit composition (40), as well as by its localization in postsynaptic structures (41), ZnT1 may endow the zinc-containing synapse with a dynamic form of regulation specific for GluN2A-containing NMDAR signals.

ZnT1 expression is also tightly coupled to fluctuations in free intracellular zinc levels (42). Rises in intracellular zinc concentrations are quickly detected by the metal regulatory element (MRE) transcription factor 1 (MTF1) to induce up-regulation of MRE-driven genes, including ZnT1 (43). As increases in intracellular zinc levels have been prominently detected following neuronal depolarization (44), it is also conceivable that the ZnT1-GluN2A complex is a key component of activity-dependent synaptic processes, perhaps even in synapses that do not express ZnT3, and, thereby, vesicular zinc. Robust NMDAR activation can lead to intracellular zinc liberation from metal-binding proteins such as metallothionein (45), independently of synaptic zinc (46), likely as a consequence of glutamate-stimulated production of oxygen-derived reactive species (47). We suggest that the observed actions of N2AZ on ZnT3-independent zinc inhibition of NMDAR-mediated responses (i.e., caged glutamate responses in cortical neurons in culture and 100-Hz stimulation of parallel fibers; Figs. 3 and 4, E to G) may be reflective of increases of intracellular zinc in response to robust NMDAR activation produced under our experimental conditions. Manipulations that enhance or diminish ZnT1 expression in cultured neurons have yielded subsequent increases or decreases in dendritic spine length, respectively (23). As NMDAR activation is a major regulator of synaptic strength and spine dynamics, ZnT1-mediated zinc inhibition may provide unique forms of synaptic plasticity through its regulation of NMDAR function.

One remaining question not fully addressed in our study is how presynaptic release of zinc and postsynaptic transport of intracellular zinc by ZnT1 cooperate to regulate NMDARs. It is tempting to assume that the source of the postsynaptic intracellular pool of zinc necessary for NMDAR block is derived from the synaptically released pool, translocating to the postsynaptic neuron. However, the predominant routes of entry for zinc (l-type calcium channels and AMPARs do not appear to contribute to zinc inhibition of NMDAR inhibition. SLC39A (Zrt- and Irt-like Proteins; ZIP) transporters, which move zinc into the cytoplasm, may serve as the route for synaptic zinc translocation, and both ZIP1 and ZIP3 have been previously observed to influence synaptic uptake of zinc, albeit under injurious conditions (48). However, further experiments will be necessary to fully assess the complex interplay between ZnT3 and ZnT1 to regulate zinc inhibition of NMDARs.

In summary, we developed a cell-permeant peptide that dissociates the zinc transporter ZnT1 from the highly zinc-sensitive NMDAR subunit GluN2A. This novel tool allowed us to uncover the mechanism by which zinc inhibits NMDAR function, which involves not only extracellular ZnT3-dependent zinc but also intracellular zinc and ZnT1-GluN2A complexes. We propose that the ZnT1-GluN2A association allows the synapse to direct zinc to its high-affinity binding site within the GluN2A-containing NMDAR by creating a physiologically and spatially distinct extracellular zinc microdomain in the synapse.

Acknowledgments: We thank C. Anderson for technical help with MATLAB and discussions and K. Hartnett-Scott for technical assistance. Funding: This work was supported by NSF grant NSF-IOS-BSF 1655480 (to E.A., T.T., and M.H.) and NIH grants DC007905 (to T.T.), NS043277 (to E.A.), GM128195 (to J.W.J.), and F31 NS1134477 (to M.B.P.). Author contributions: R.F.K., E.A., T.T., A.M., R.K., H.A., M.H., M.B.P., and J.W.J. designed experiments. R.F.K., A.M., H.A., and M.B.P. conducted experiments and performed data analysis. R.F.K., E.A., and T.T. wrote the manuscript. Competing interests: A provisional patent application entitled Peptides that enhance NMDA receptor function and use thereof, application no. 62/942,979, was filed on 3 December 2019 (assignee: University of Pittsburgh; co-inventors: E.A. and T.T.). The authors declare no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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