Hydrophobic interactions of n-alkyl diamines with the N-methyl-D-aspartate receptor: Voltage-dependent and -independent blocking sites

Swaminathan Subramaniam, Sean D. Donevan, Michael A Rogawski

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31 Scopus citations


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 languageEnglish (US)
Pages (from-to)117-124
Number of pages8
JournalMolecular Pharmacology
Issue number1
StatePublished - Jan 1994
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology


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