We examined the block of N-methyl-D-aspartate (NMDA) receptors by n-alkyl (straight chain) diamines and related monoamines and triamines using whole- cell voltage clamp recording of NMDA receptor currents in cultured rat hippocampal neurons and [3H] dizocilpine binding to rat forebrain homogenates. At -60 mV, the diamines (carbon chain lengths 3-12) produced a concentration-dependent inhibition of NMDA receptor current (IC50 values, 6128-7.3 μM). For diamines of carbon chain lengths greater than 6, the inhibition was partially, but not completely, relieved by depolarization, indicating that the block occurs at distinct voltage-dependent and voltage- independent sites. The block produced by short-chain diamines (carbon chain lengths 3-6) was completely relieved by depolarization, indicating little or no interaction with the voltage-independent site. In comparison with the corresponding diamines, homologous monoamines exhibited very low potency, whereas homologous triamines were of equal or lower potency. For long-chain diamines, inhibitory potency at both the voltage-dependent and voltage- independent sites was correlated with carbon chain length (binding energy increasing 600-700 cal/mol-CH2), suggesting that binding to each of the sites is stabilized by a hydrophobic interaction. Affinities for the voltage- dependent blocking site (transformed to 0 mV) and for the voltage-independent blocking site were similar. These values were also similar to the inhibitory potencies of the diamines in the [3H]dizocilpine binding assay. Analysis of the voltage-dependence of block at the voltage-dependent site yielded zδ values for diamines of intermediate length (carbon chain lengths 7-9) that decreased with increasing length from 0.91 to 0.63 [approaching the zδ values of monovalent blockers (~0.54) and one-half of the zδ values of shorter diamines (~1.1)], suggesting that the intermediate length diamines block in a linear, extended chain conformation with one of the charges having incomplete access to a deep binding site. Longer chain diamines (carbon chain lengths 10 and 12) exhibited larger zδ values (0.78 and 0.98, respectively), presumably because enhanced conformational flexibility permitted a folded- over conformation. From the intercharge distances of the intermediate length diamines in their lowest energy conformation, we estimated that the total voltage drop within the NMDA receptor channel occurs over a distance of ~20 Å. The putative polyamine facilitatory site antagonist diethylenetriamine inhibited NMDA-induced currents at the voltage-dependent site (IC50, 654 μM; -60 mV). However, at positive potentials, diethylenetriamine neither produced block by itself nor reversed the inhibitory effect of diamines, indicating that it is not an antagonist at the voltage-independent blocking site. We conclude that the NMDA receptor-complex possesses two distinct hydrophobic diamine blocking sites, one of which is voltage-dependent and the other which is not. The available evidence suggests that these sites are distinct from the site at which polyamines such as spermine allosterically facilitate channel opening.
|Original language||English (US)|
|Number of pages||8|
|State||Published - Jan 1994|
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