Zinc inhibition of rat NR1/NR2A N-methyl-d-aspartate receptors

Zinc ions (Zn2+) are localized in presynaptic vesicles at glutamatergic synapses and released in an activity-dependent manner. Modulation of NMDA-type glutamate receptors by extracellular Zn2+ may play an important role under physiological conditions and during pathologies such as ischaemia or seizure. Zn2+ inhibits NMDA receptors containing the NR2A subunit with an IC50 value in the low nanomolar concentration range. Here we investigate at the single-channel level the mechanism of high affinity Zn2+ inhibition of recombinant NR1/NR2A receptors expressed in HEK293 cells. Zn2+ reversibly decreases the mean single-channel open duration and channel open probability determined in excised outside-out patches, but has no effect on single-channel current amplitude. A parallel series of experiments demonstrates that lowering extracellular pH (increasing proton concentration) has a similar effect on NR1/NR2A single-channel properties as Zn2+. Fitting the sequence of single-channel events with kinetic models suggests that the association of Zn2+ with its binding site enhances proton binding. Modelling further suggests that protonated channels are capable of opening but with a lower open probability than unprotonated channels. These data and analyses are consistent with Zn2+-mediated inhibition of NMDA receptors primarily reflecting enhancement of proton inhibition

F-00162-2023R1 SUPPLEMENTAL FIGURES <b>Role of protease activated receptor 4 (PAR4) in mouse models of acute </b><b>and chronic </b><b>kidney injury</b>.pdf

SUPPLEMENTAL FIGURE 1. Top: PAR4 antibody (clone 14H6) Western Blot for PAR4 tetracylcine inducible TRex cells comparing – or + tetracycline. Bottom: The same blot stripped and reprobed for actin as a loading control.SUPPLEMENTAL FIGURE 2. Immunofluorescent staining shows similar renal macrophage (CD68) and kidney TNF-α expression in WT and PAR4KO mice 7 days after UUO. Scale bar=50μm.</p

Modulation of glycine potency in rat recombinant NMDA receptors containing chimeric NR2A/2D subunits expressed in Xenopus laevis oocytes

Heteromeric NMDARs are composed of coagonist glycine-binding NR1 subunits and glutamate-binding NR2 subunits. The majority of functional NMDARs in the mammalian central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there are four types (A–D). We show that the potency of a variety of endogenous and synthetic glycine-site coagonists varies between recombinant NMDARs such that the highest potency is seen at NR2D-containing and the lowest at NR2A-containing NMDARs. This heterogeneity is specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged the S1 and S2 regions that form the ligand-binding domains and coexpressed these with NR1 subunits in Xenopus laevis oocytes. Glycine concentration–response curves for NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC50 values similar to NR2A(WT)-containing NMDARs. However, receptors containing NR2A subunits including the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen in NR2D(WT)-containing NMDARs. In particular, two residues in the S2 region of the NR2A subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D subunit influence glycine potency. We conclude that the variation in glycine potency is caused by interactions between the NR1 and NR2 ligand-binding domains that occur following agonist binding and which may be involved in the initial conformation changes that determine channel gating

Allosteric interaction between zinc and glutamate binding domains on NR2A causes desensitization of NMDA receptors

Fast desensitization is an important regulatory mechanism of neuronal NMDA receptor function. Previous work suggests that fast desensitization of NR1/NR2A receptors is caused by ambient zinc, and that a positive allosteric interaction occurs between the extracellular zinc-binding amino terminal domain and the glutamate-binding domain of NR2A. The relaxation of macroscopic currents in the presence of zinc reflects a shift to a new equilibrium due to increased zinc affinity following the binding of glutamate. Here we demonstrate that this allosteric coupling reflects interactions within the NR2A subunit, and that the affinity of zinc for its binding site is regulated by glutamate binding and not by glycine binding nor by channel pore opening. We fit an explicit model to experimental data over a wide range of parameters, demonstrating that allosteric theory can quantitatively account for the fast zinc-dependent component of desensitization for NR1/NR2A NMDA receptors. We subsequently use this model to evaluate the effects of extracellular zinc on NR1/NR2A excitatory postsynaptic currents (EPSCs) by simulating the response to a brief synaptic-like pulse of glutamate. Modelling results show that zinc at a steady-state concentration of at least 100 nm has a significant effect on the amplitude of NMDA EPSCs but that concurrent release of 10 μm zinc with synaptic glutamate release has little effect on the amplitude of a single NR1/NR2A NMDA EPSC. These data suggest that while steady-state zinc can regulate the amplitude of synaptic NMDA currents, zinc co-released with glutamate will only have significant impact under conditions of high frequency activity or at concentrations high enough to cause voltage-dependent channel block

Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux

The SNARE protein syntaxin1A (SYN1A) interacts with and regulates the function of transmembrane proteins including ion channels and neurotransmitter transporters. Here we define the first 33 amino acids of the N-terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch clamp technique with amperometry we measured DA release under voltage clamp. At −60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 µM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at −60 mV both in hDAT cells and DA neurons. Recently, it has been shown that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity re-uptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural, and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is a required for substrate efflux. In Drosophila melanogaster, expression of hDAT T356M in DA neurons lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions

Rare Autism-Associated Variants Implicate Syntaxin 1 (STX1 R26Q) Phosphorylation and the Dopamine Transporter (hDAT R51W) in Dopamine Neurotransmission and Behaviors

Background: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. Methods: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD). Outcomes: Here, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis. Interpretation: We determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD