We investigated the mechanisms by which the antiparkinsonian and neuroprotective agents amantadine and memantine inhibit responses to N-methyl-D-aspartic acid (NMDA). Whole cell recordings were performed using cultured rat cortical neurons or Chinese hamster ovary (CHO) cells expressing NMDA receptors. Both amantadine and memantine blocked NMDA-activated channels by binding to a site at which they could be trapped after channel closure and agonist unbinding. For neuronal receptors, the IC50s of amantadine and memantine at -67 mV were 39 and 1.4 microM, respectively. When memantine and agonists were washed off after steady-state block, one-sixth of the blocked channels released rather than trapped the blocker; memantine exhibited "partial trapping." Thus memantine appears to have a lesser tendency to be trapped than do phencyclidine or (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[1,d]cyclihepten-5,1 0-imine (MK-801). We next investigated mechanisms that might underlie partial trapping. Memantine blocked and could be trapped by recombinant NMDA receptors composed of NR1 and either NR2A or NR2B subunits. In these receptors, as in the native receptors, the drug was released from one-sixth of blocked channels rather than being trapped in all of them. The partial trapping we observed therefore was not due to variability in the action of memantine on a heterogeneous population of NMDA receptors in cultured cortical neurons. Amantadine and memantine each noncompetitively inhibited NMDA-activated responses by binding at a second site with roughly 100-fold lower affinity, but this form of inhibition had little effect on the extent to which memantine was trapped. A simple kinetic model of blocker action was used to demonstrate that partial trapping can result if the presence of memantine in the channel affects the gating transitions or agonist affinity of the NMDA receptor. Partial trapping guarantees that during synaptic communication in the presence of blocker, some channels will release the blocker between synaptic responses. The extent to which amantadine and memantine become trapped after channel block thus may influence their therapeutic effects and their modulation of NMDA-receptor-mediated excitatory postsynaptic potentials.

The sodium channel initiates action potentials by opening in response to membrane depolarization. Fast channel inactivation, which is required for proper physiological function, is mediated by a cytoplasmic loop proposed to occlude the ion pore via a hinged lid mechanism with the triad IFM serving as a hydrophobic "latch". The NMR solution structure of the isolated inactivation gate reveals a stably folded core comprised of an alpha-helix capped by an N-terminal turn, supporting a model in which the tightly folded core containing the latch motif pivots on a more flexible hinge region to occlude the pore during inactivation. The structure, in combination with substituted cysteine mutagenesis experiments, indicates that the IFM triad and adjacent Thr are essential components of the latch and suggests differing roles for the residues of the IFMT motif in fast inactivation.

The N-methyl-D-aspartate (NMDA) receptor is a ligand-gated ion channel involved in excitatory neurotransmission, synaptic plasticity, and neuronal cell death. Two families of NMDA receptor subunits exist: the NR1 subunit (1), which is found in eight alternatively spliced isoforms (2), and the NR2 subunit, for which four subtypes have been described (NR2A-D), each one being a different gene product (3-7). Functional NR1 homomers apparently can be assembled in frog oocytes (1), whereas receptors in mammalian cells likely are formed by the coassembly of the NR1 subunit with at least one type of NR2 subunit (8-13). Although it recently has been suggested that functional NMDA channels most likely contain two NR1 subunits (ref. 14, but see ref. 15), the number of NR2 subunits that coassemble with NR1 has yet to be determined.Individual NR2 subunits coassembling with NR1 produce recombinant NMDA receptors with varying sensitivity to pharmacological agents (16-19) and different channel permeation and kinetic properties (4,6,(20)(21)(22)(23)(24). Hence, the number and type of NR2 subunits in a single receptor likely will influence its overall function. In the present study, we have developed a series of profiles, based on unique biophysical and pharmacological properties, for four different combinations of NR1, NR2A, NR2B, and NR2C subunits transiently expressed in Chinese hamster ovary (CHO) cells (25).

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