Zinc Effects on NMDA Receptor Gating Kinetics

Abstract

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.

Inhibition of GluN2A-Containing N-Methyl-d-Aspartate Receptors by 2-Naphthoic Acid

“…By fitting this model to single-channel data we determined that the change in NPA affinity between closed and open states is ∼5-fold, with NPA dissociation rate being 5-fold faster from open relative to closed receptors. However, given that this statedependency is relatively weak, with the change in free-energy being on the same scale with those previously reported for other NMDA receptor modulators (Amico-Ruvio et al, 2011, and also with the difference in free energy between N2A and N2B receptors Amico-Ruvio and Popescu, 2010), we anticipate that the magnitude of the NPA effects will vary substantially with experimental conditions that include such modulators and will also vary with the NMDA receptor isoform considered.…”

Ifenprodil Effects on GluN2B-Containing Glutamate Receptors

“…On the basis of these analyses, we conclude that ifenprodil-bound receptors had shorter openings on average because they spent more time in the low-activity gating mode; however, medium-and high-activity modes were still accessible and during these periods, openings were of a duration similar to those of ifenprodil-free receptors. This inhibitory mechanism was unexpected because it was not observed with zinc, which is also an NTD ligand (Amico-Ruvio et al, 2011).…”

“…The recording pipette containing the excised membrane patch was positioned within one stream close to the interface, and the theta tube was mounted on a piezoelectric translator (Burleigh LSS-3100/3200) and used to exchange solution around the patch pipette. Open-tip potential measurements done at the end of each experiment showed that the 10 to 90% exchange occurred within 0.15 to 0.25 ms (Amico-Ruvio et al, 2011). Each barrel of the theta tube was connected to several solutions through a micro-manifold controlled with pinch valves (VC 6; Warner Instruments, Hamden, CT).…”

“…Furthermore, as mathematical descriptions of single-molecule behaviors have continued to improve, detailed kinetic mechanisms for these inhibitory actions have been delineated Amico-Ruvio et al, 2011). Essentially, the activation of 2B receptors consists of rapid agonist binding, slow channel gating, even slower channel desensitization, and occasional gatingmode changes.…”

Experimental Modelling of Spinal Anaesthesia in Rats

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