Trapping Channel Block of NMDA-Activated Responses By Amantadine and Memantine

Abstract

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.

Amantadine Inhibits NMDA Receptors by Accelerating Channel Closure during Channel Block

“…This K d is considerably larger than the amantadine IC 50 (38.9 Ϯ 4.6 M) that we measured under similar conditions in whole-cell experiments (Blanpied et al, 1997), a value in agreement with the average amantadine IC 50 (ϳ35 M) measured electrophysiologically by others (Parsons et al, 1995(Parsons et al, , 1999aSobolevsky and Yelshansky, 2000;Bolshakov et al, 2003). The observation that K d Ͼ IC 50 implies that amantadine inhibits total NMDA receptor-mediated current more effectively than it blocks open single channels.…”

“…For example, blockers that act by the "sequential" (or "foot-in-the-door") mechanism, which prevent closure of the channel while blocking, inhibit whole-cell responses (or patch n ϫ P open ) much less effectively than they block single-channel currents (K d Ͻ Ͻ IC 50 ) (Neher and Steinbach, 1978;Hille, 2001;Johnson and Qian, 2002). Amantadine, in contrast, is known to be a trapping channel blocker: it permits channel closure while bound in the channel of NMDA receptors (Blanpied et al, 1997;Sobolevsky and Yelshansky, 2000;Bolshakov et al, 2003). Nevertheless, even trapping channel blockers have been found to partly inhibit channel closure during block , leading to an IC 50 that is greater than the K d .…”

“…Its antiviral action results from block of the M2 small viral membrane protein (Hay, 1992). Although the utility of amantadine in treating Parkinson's disease was initially assumed to result from effects on dopaminergic systems (Danysz et al, 1997), amantadine later was found to block the channel of NMDA receptors (Kornhuber et al, 1991;Lupp et al, 1992;Parsons et al, 1995;Blanpied et al, 1997). Increasing evidence suggests that the effectiveness of amantadine in treating nervous system disorders results predominantly from its inhibition of NMDA responses (Blanchet et al, 2003).…”

“…Whole-cell recordings from cultured neurons or transfected HEK 293T cells were performed as described previously (Blanpied et al, 1997), using the same solutions as for outside-out patch recordings. Rapid applications of the indicated NMDA concentration plus 10 M glycine with or without amantadine were accomplished by moving a set of gravity-fed flow pipes under computer control.…”

“…Rapid applications of the indicated NMDA concentration plus 10 M glycine with or without amantadine were accomplished by moving a set of gravity-fed flow pipes under computer control. Solution exchanges were at least 98% complete within 120 ms (Blanpied et al, 1997).…”