Blocker studies of the functional architecture of the NMDA receptor channel.

Blockade of ion channels passing through the NMDA receptors of isolated rat hippocampus pyramidal neurons with tetraalkylammonium compounds, 9-aminoacridine, and Mg2+ was studied using patch-clamp methods in the whole-cell configuration. Currents through NMDA channels were evoked by application of 100 microM aspartate in magnesium-free medium containing glycine (3 microM) to neurons. Analysis of the kinetics, charge transfer, and relationships between the extent of suppression of stationary currents on the one hand and membrane potential, agonist concentration, and blocker concentration on the other showed that blockers had different effects on the closing, desensitization, and agonist dissociation of NMDA channels. The size of the blocker was found to be the decisive factor determining its action on the gating functions of NMDA channels: larger blockers prevented closure and/or desensitization of the channel; smaller blockers only had partial effects on these processes, while the smallest blockers had no effect at all. These experiments showed that the apparent affinity of the blocker for the channel (1/IC50) depended not only on the microscopic equilibrium dissociation constant (Kd), but also on the number of blocker binding sites, their mutual influences, and, of particular importance, the interaction of the blocker with the gating structures of the channel. These data led us to propose hypotheses relating to the geometry of the NMDA channel and the structure of its gating mechanism. The channel diameter at the level of activated gates was estimated to be 11 A.

[Effect of the potential antiparkinsonian agent adamantane derivative on ion channels of NMDA glutamate receptors]. / Vzaimodeistvie potentsial'nogo protivoparkinsonicheskogo sredstva proizvodnogo adamantana s ionnymi kanalami glutamatnykh retseptorov NMDA podtipa.

Interaction of a new potential antiparkinsonian drug, N-(2-adamantyl)hexamethyleneimine hydrochloride (A-7), with NMDA channels in acutely isolated rat hippocampal neuron culture was studied by the whole-cell patch-clamp technique. The currents through the NMDA channels were excited by aspartate (Asp) application in a magnesium-free glycine-containing (3 microM) medium. It was found that A-7 produced a concentration-dependent (IC50 = 11.8 +/- 0.6 microM) NMDA channel blocking. The blocking rate increased with the A-7 concentration, whereas the unblocking was concentration-independent. The degree of blocking was independent of the Asp concentration, but was markedly increased by hyperpolarization of the cell membrane. After Asp washout, A-7 remained trapped in the channel by the activation gate. The channel was unblocked upon the next Asp application. These data is evidence that the antiparkinsonian effect of A-7 is related to the NMDA-channel-blocking activity of the drug.

[Study functional architecture of NMDA receptor channels with their blockers]. / Izuchenie funktsional'noi arkhitektury kanala NMDA-receptorov s pomoshch'iu blokatorov.

Blockade of ionic currents through NMDA receptor channels in acutely isolated rat hippocampal neurons by tetraalkylammonium compounds, 9-aminoacridine and Mg2+ was studied using whole-cell patch-clamp technique. The currents through NMDA channels were elicited by 100 microM aspartate application in a Mg(2+)-free 3 microM glycine-containing solution. An analysis of the kinetics, charge transfer and dependencies of the stationary current inhibition on the membrane potential and the agonist and the blocker concentrations showed that the blockers affect NMDA channel closure, desensitization and the agonist dissociation in different ways. The size of the blocker proved to be the determinant of the blocker action on the NMDA channel gating machinery: large blockers prevented the channel closure and/or desensitization, smaller ones only partly affected these processes, while the smallest did not affect at all. It was shown that the apparent blocker affinity to the channel, 1/IC50, depended not only on the microscopic dissociation constant, Kd, but also on the number of the blocker binding sites, their mutual dependence, and, which is much more important, on the blocker interaction with the channel gating machinery. Based upon the data obtained, there was advanced hypotheses on the NMDA channel geometry and the structure of its gating machinery. The diameter of the channel at the level of the activation gate was estimated as 11 A.