NMDA receptors are heteromeric glutamate-gated channels composed of GluN1 and GluN2 subunits. Receptor isoforms that differ in their GluN2-subunit type (A-D) are expressed differentially throughout the central nervous system and have distinct kinetic properties in recombinant systems. How specific receptor isoforms contribute to the functions generally attributed to NMDA receptors remains unknown, due in part to the incomplete functional characterization of individual receptor types and unclear molecular composition of native receptors. We examined the stationary gating kinetics of individual rat recombinant GluN1/GluN2B receptors in cell-attached patches of transiently transfected HEK293 cells and used kinetic analyses and modeling to describe the full range of this receptor's gating behaviors. We found that, like GluN1/GluN2A receptors, GluN1/GluN2B receptors have three gating modes that are distinguishable by their mean open durations. However, for GluN1/GluN2B receptors, the modes also differed markedly in their mean closed durations and thus generated a broader range of open probabilities. We also found that regardless of gating mode, glutamate dissociation occurred approximately 4-fold more slowly (k(-) = 15 s(-1)) compared to that observed in GluN1/GluN2A receptors. On the basis of these results, we suggest that slow glutamate dissociation and modal gating underlie the long heterogeneous activations of GluN1/GluN2B receptors.

Zinc accumulates in the synaptic vesicles of certain glutamatergic forebrain neurons and modulates neuronal excitability and synaptic plasticity by multiple poorly understood mechanisms. Zinc directly inhibits NMDA-sensitive glutamate-gated channels by two separate mechanisms: high-affinity binding to N-terminal domains of GluN2A subunits reduces channel open probability, and low-affinity voltage-dependent binding to pore-lining residues blocks the channel. Insight into the high-affinity allosteric effect has been hampered by the receptor's complex gating; multiple, sometimes coupled, modulatory mechanisms; and practical difficulties in avoiding transient block by residual Mg(2+). To sidestep these challenges, we examined how nanomolar zinc concentrations changed the gating kinetics of individual block-resistant receptors. We found that block-insensitive channels had lower intrinsic open probabilities but retained high sensitivity to zinc inhibition. Binding of zinc to these receptors resulted in longer closures and shorter openings within bursts of activity but had no effect on interburst intervals. Based on kinetic modeling of these data, we conclude that zinc-bound receptors have higher energy barriers to opening and less stable open states. We tested this model for its ability to predict zinc-dependent changes in macroscopic responses and to infer the impact of nanomolar zinc concentrations on synaptic currents mediated by 2A-type NMDA receptors.

Background: The C-terminal domains (CTDs) of NMDA receptors are essential for normal brain function. Results: We developed kinetic mechanisms for receptors lacking CTDs using single-channel methods. Conclusion: GluN1 CTDs control primarily unitary conductance and GluN2 CTDs control gating kinetics. Significance: Results afford quantitative insight into how intracellular perturbations can change the time course of NMDA receptor currents.

BackgroundDespite advances in the treatment of primary breast tumors, the outcome of metastatic breast cancer remains dismal. Brain metastases present a particularly difficult therapeutic target due to the “sanctuary” status of the brain, with resulting inability of most chemotherapeutic agents to effectively eliminate cancer cells in the brain parenchyma. A large number of breast cancer patients receive various neuroactive drugs to combat complications of systemic anti-tumor therapies and to treat concomitant diseases. One of the most prescribed groups of neuroactive medications is anti-depressants, in particular selective serotonin reuptake inhibitors (SSRIs). Since SSRIs have profound effects on the brain, it is possible that their use in breast cancer patients could affect the development of brain metastases. This would provide important insight into the mechanisms underlying brain metastasis. Surprisingly, this possibility has been poorly explored.MethodsWe studied the effect of fluoxetine, an SSRI, on the development of brain metastatic breast cancer using MDA-MB-231BR cells in a mouse model.ResultsThe data demonstrate that fluoxetine treatment increases the number of brain metastases, an effect accompanied by elevated permeability of the blood–brain barrier, pro-inflammatory changes in the brain, and glial activation. This suggests a possible role of brain-resident immune cells and glia in promoting increased development of brain metastases.ConclusionOur results offer experimental evidence that neuroactive substances may influence the pathogenesis of brain metastatic disease. This provides a starting point for further investigations into possible mechanisms of interaction between various neuroactive drugs, tumor cells, and the brain microenvironment, which may lead to the discovery of compounds that inhibit metastasis to the brain.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-598) contains supplementary material, which is available to authorized users.

N-Methyl-D-aspartate (NMDA) receptors are glutamate-and glycine-gated channels that mediate fast excitatory transmission in the central nervous system and are critical to synaptic development, plasticity, and integration. They have a rich complement of modulatory sites, which represent important pharmacological targets. Ifenprodil is a well tolerated NMDA receptor inhibitor; it is selective for GluN2B-containing receptors and has neuroprotective effects. The mechanism by which ifenprodil inhibits NMDA receptor responses is not fully understood. The inhibition is incomplete and noncompetitive with other known NMDA receptor agonists or modulators, although reciprocal effects have been reported between ifenprodil potency and that of extracellular ligands including glutamate, glycine, zinc, protons, and polyamines. Recent structural studies revealed that ifenprodil binds to a unique site at the interface between the extracellular N termini of GluN1 and GluN2B subunits, supporting the view that interactions with other extracellular modulators are indirect. In this study, we examined how ifenprodil affects the gating reaction of NMDA receptors in conditions designed to minimize actions by contemporaneous ligands. We found that ifenprodil decreased NMDA receptor equilibrium open probability by raising an energetic barrier to activation and also by biasing the receptor toward low open probability gating modes. These results demonstrate intrinsic effects of ifenprodil on NMDA receptor stationary gating kinetics and provide means to anticipate how ifenprodil will affect receptor responses in defined physiological and pathological circumstances.