HYDROPHOBIC INTERACTIONS OF N-ALKYL DIAMINES WITH THE N-METHYL-D-ASPARTATE RECEPTOR - VOLTAGE-DEPENDENT AND VOLTAGE-INDEPENDENT BLOCKING SITES

We examined the block of N-methyl-D-aspartate (NMDA) receptors by n-al kyl (straight chain) diamines and related monoamines and triamines usi ng whole-cell voltage clamp recording of NMDA receptor currents in cul tured rat hippocampal neurons and [H-3] dizocilpine binding to rat for ebrain homogenates. At -60 mV, the diamines (carbon chain lengths 3-12 ) produced a concentration-dependent inhibition of NMDA receptor curre nt (IC50 values, 6128-7.3 mu M). For diamines of carbon chain lengths greater than 6, the inhibition was partially, but not completely, reli eved by depolarization, indicating that the block occurs at distinct v oltage-dependent and voltage-independent sites. The block produced by short-chain diamines (carbon chain lengths 3-6) was completely relieve d by depolarization, indicating little or no interaction with the volt age-independent site. In comparison with the corresponding diamines, h omologous monoamines exhibited very low potency, whereas homologous tr iamines were of equal or lower potency. For long-chain diamines, inhib itory potency at both the voltage-dependent and voltage-independent si tes 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-d ependent blocking site (transformed to 0 mV) and for the voltage-indep endent blocking site were similar. These values were also similar to t he inhibitory potencies of the diamines in the [H-3]dizocilpine bindin g assay. Analysis of the voltage-dependence of block at the voltage-de pendent site yielded z delta values for diamines of intermediate lengt h (carbon chain lengths 7-9) that decreased with increasing length fro m 0.91 to 0.63 [approaching the z delta values of monovalent blockers (similar to 0.54) and one-half of the z delta values of shorter diamin es (similar to 1.1)], suggesting that the intermediate length diamines block in a linear, extended chain conformation with one of the charge s having incomplete access to a deep binding site. Longer chain diamin es (carbon chain lengths 10 and 12) exhibited larger z delta values (0 .78 and 0.98, respectively), presumably because enhanced conformationa l flexibility permitted a folded-over conformation. From the interchar ge distances of the intermediate length diamines in their lowest energ y conformation, we estimated that the total voltage drop within the NM DA receptor channel occurs over a distance of similar to 20 Angstrom. The putative polyamine facilitatory site antagonist diethylenetriamine inhibited NMDA-induced currents at the voltage-dependent site (IC50, 654 mu M; -60 mV). However, at positive potentials, diethylenetriamine neither produced block by itself nor reversed the inhibitory effect o f diamines, indicating that it is not an antagonist at the voltage-ind ependent blocking site. We conclude that the NMDA receptor-complex pos sesses two distinct hydrophobic diamine blocking sites, one of which i s voltage-dependent and the other which is not. The available evidence suggests that these sites are distinct from the site at which polyami nes such as spermine allosterically facilitate channel opening.