Block of the channel of N-methyl-D-aspartate (12,(19)(20)(21)(22). The M2 region is a pore loop that enters and exits the membrane intracellularly and is believed to form a large portion of the channel of NMDA receptors. A physical location of the Mg o 2ϩ -blocking site near the external tip of the M2 region appears inconsistent with an electrical location near the internal extreme of the channel.One mechanism by which the voltage dependence of block by -binding sites deep in the channel (19) and on the external side of the channel (10, 27) and for permeant monovalent cation-binding sites on both sides of the channel (26). We recently demonstrated that occupation of the permeant monovalent cation-binding sites has a profound effect on the blocking kinetics of two adamantane derivatives that block the channel of NMDA receptors (26). Here, we examine how occupation of these permeant monovalent cation-binding sites influences block by Mg o 2ϩ . Experimental ProceduresPreparation and Solutions. Primary cultures of rat cortical neurons were prepared from 16-day embryos as described (28). Neurons were used for experiments after 15-30 days in culture.Recordings of single-channel currents were performed by using outside-out patches (29) at room temperature (21-23°C). Pipettes were filled with one of three solutions. The control (130

1. The mechanisms by which four adamantane derivatives (IEM-1857(IEM- , -1592(IEM- , -1460(IEM- and -1754 block the open NMDA-activated channel were studied at membrane voltages (Vm) from -170 to +30 mV. The rate constants of channel block (k+) and of channel unblock (k_) were measured from the fully resolvable flicker of single-channel currents induced by each compound.

Using a fluorescent viability assay, immunocytochemistry, patch-clamp recordings, and Ca 2ϩ imaging analysis, we report that ouabain, a specific ligand of the Na ϩ ,K ϩ -ATPase cardiac glycoside binding site, can prevent glutamate receptor agonistinduced apoptosis in cultured rat cortical neurons. In our model of excitotoxicity, a 240-min exposure to 30 M N-methyl-Daspartate (NMDA) or kainate caused apoptosis in ϳ50% of neurons. These effects were accompanied by a significant decrease in the number of neurons that were immunopositive for the antiapoptotic peptide Bcl-2. Apoptotic injury was completely prevented when the agonists were applied together with 0.1 or 1 nM ouabain, resulting in a greater survival of neurons, and the percentage of neurons expressing Bcl-2 remained similar to those obtained without agonist treatments. In addition, subnanomolar concentrations of ouabain prevented the increase of spontaneous excitatory postsynaptic current's frequency and the intracellular Ca 2ϩ overload induced by excitotoxic insults. Loading neurons with 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid or inhibition of the plasma membrane Na ϩ ,Ca 2ϩ -exchanger by 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate (KB-R7943) eliminated ouabain's effects on NMDA-or kainite-evoked enhancement of spontaneous synaptic activity. Our data suggest that during excitotoxic insults ouabain accelerates Ca 2ϩ extrusion from neurons via the Na ϩ ,Ca 2ϩ exchanger. Because intracellular Ca 2ϩ accumulation caused by the activation of glutamate receptors and boosted synaptic activity represents a key factor in triggering neuronal apoptosis, up-regulation of Ca 2ϩ extrusion abolishes its development. These antiapoptotic effects are independent of Na ϩ ,K ϩ -ATPase ion transport function and are initiated by concentrations of ouabain that are within the range of an endogenous analog, suggesting a novel functional role for Na ϩ ,K ϩ -ATPase in neuroprotection.

We report the presence of binding sites for permeant monovalent cations at the internal and external entrances to the channel of NMDA receptors. We measured the effects of changing internal cesium (Cs+) and external sodium (Na+) concentrations on the channel-blocking kinetics of the adamantane derivatives IEM-1754 and IEM-1857. Binding of Na+, or of Cs+ after it permeates the channel, to sites at the external channel entrance prevents blockers from entering the channel. Binding of Na+ to a blocked channel prevents blocker unbinding. Cs+ binding to a site at the internal channel entrance prevents IEM-1754 from occupying the deeper of its two sites of block. The results show the critical effects of permeant ions on the kinetics, affinity and voltage-dependence of channel blockers.