Xenon blocks AMPA and NMDA receptor channels by different mechanisms.

The noble gas xenon (Xe) inhibits not only NMDA receptors (NMDARs) but also the two other subtypes of glutamate receptor i.e. AMPA (AMPARs) and kainate receptors. Preliminary studies on AMPARs suggest that Xe sensitivity might be coupled to receptor desensitization. In order to find out if this hypothesis can be applied to all glutamate receptors, we analyzed additional 'non-desensitizing' AMPARs mutants and compared these with homologous mutants of NMDARs. Membrane currents of Neuro2A or SH-SY5Y cells transfected with cDNA encoding AMPA- or NMDA receptors were investigated by whole cell recordings under voltage clamp conditions. Agonists (glutamate, kainate, NMDA) were applied to the cells by means of a rapid perfusion system. Xenon was preincubated for 20 s before testing it in combination with the particular agonist. Xe (3.5 mM) reduced peak and plateau currents of AMPA wild-type receptors [GluR1(i); GluR2(i,Q)] activated for 5 s with 3 mM glutamate, by 45 and 55% respectively. With mutant AMPARs showing greatly diminished or abolished desensitization i.e. GluR1(i)_L497Y, GluR1(i)_A636T(Lc) GluR2(i,Q)_R649E and GluR2(i,Q)_A643T(Lc) the reduction by Xe was significantly smaller and varied by between 4 and 20%. In contrast, no difference in the blocking capacity of Xe was observed comparing wild-type NR1-1a/NR2A receptors with receptors having point mutations within NR2A that substantially slowed (NR2A_A651T(Lc)) or accelerated (NR2A_M823W) receptor desensitization. Thus, our data indicate that in AMPARs channel blockade by Xe is related to desensitization, whereas in NMDARs no evidence for such a relation was found. Thus, Xe seems to exert its inhibiting effect on various ionotropic glutamate receptors by different molecular mechanisms.

Lipid emulsions reduce NMDA-evoked currents.

Membrane currents conducted by the NMDA receptor channels were investigated in cultured cortical neurons and TsA cells transfected with NR1-1a/NR2A subunits of the NMDA receptor. The whole-cell recording technique was used. Current transients evoked by bath application of NMDA for 5 s were characterized by a fast peak and a slow decay to 46.1 +/-15.5% of the peak level at the end. When NMDA was applied in combination with various lipid emulsions (Intralipid, ClinOleic, Lipofundin or Abbolipid, the NMDA-induced currents were reduced, although this reduction did not affect the fast peak, it did affect the decay phase. The amount of reduction depended on the concentration of the lipids (in the case of Abbolipid diluted at 1:40, the current at the end of the 5-s drug application was approximately 2/3 of control). When Abbolipid was applied 40 s before NMDA, peak and late current were reduced to approximately 2/3. The effect of current reduction was the same at either of the two chosen membrane potentials (-80 and +40 mV) which indicates that the effect was not mediated by contamination of the emulsions with Mg(2+). The current reduction produced by Abbolipid was about the same in native neuronal cells and in TsA cells expressing the NR1-1a/NR2A subunits. The current-reducing effect of the lipid emulsions may add to the anesthetic, analgesic and neuroprotective effects seen with hypnotics administered by way of lipid carriers.

Evidence that Xenon does not produce open channel blockade of the NMDA receptor.

Previous studies had not excluded the possibility that the mechanism by which Xenon (Xe) blocks N-methyl-D-aspartate (NMDA) receptors might be that of an open-channel blocker. We tested this possibility on mutant NMDA receptors carrying an alanine (A) to cysteine (C) mutation located within the SYTANLAAF-motif of the third transmembrane region (TM3). This mutation was shown to yield constitutively open ion channels after modification with a thiol-modifying reagent. We expressed such mutant channels in Neuro2A cells and recorded glutamate (50 microM)-induced currents in the whole cell recording mode. Although Xe (3.5 mM) blocked the currents through the wild-type receptor NR1-1a/NR2A and NR1-1a/NR2B by approximately 40% and those through the mutant receptors NR1-1a/NR2A(A650C) or NR1-1a/NR2B(A651C) by approximately 30%, it was unable to block the currents through the methane thiosulfonate etyhlammonium-modified mutant receptors. On the other hand, established open-channel blockers of the NMDA receptor such as MK-801 (1 microM) or Mg ions (Mg(2+); 1 mM) were able to block these permanently open channels. These results suggest that Xe does not act as a classical open-channel blocker at the NMDA receptor.