Abstract

The purpose of this study was to characterize the kinetics and voltage-dependence of the block ofN-methyl-d-aspartate (NMDA)-induced currents in primary cultures of rat cortical neurons by the neuroprotective, low-affinity, NMDA antagonist ARL 15896AR, using whole-cell voltage-clamp techniques. ARL 15896AR caused rapid and reversible inhibition of NMDA (50 μM)-evoked currents from neurons held at −60 mV, with an IC₅₀ of 9.8 μM. The EC₅₀ for NMDA was not significantly affected by 10 μM ARL 15896AR (P > .05), consistent with a noncompetitive mechanism of block. ARL 15896AR antagonism was use-dependent, because application of the drug 60 sec before NMDA did not attenuate the initial NMDA-evoked current, although the block developed rapidly thereafter. Once bound, ARL 15896AR remained trapped upon removal of NMDA until subsequent NMDA re-exposure, whereupon currents recovered rapidly. The forward and reverse binding rate constants were estimated to be 2.406 × 10⁴ M⁻¹ sec⁻¹ and 0.722 sec⁻¹, respectively. Antagonism was strongly voltage-dependent; the K _D values at 0 and −60 mV were 60 and 11 μM, respectively. Additionally, there was a component of the block by ARL 15896AR that was voltage-insensitive. This component of the block did not act at the ligand binding site, because it was not influenced by NMDA concentration, or at the polyamine site, because it was not affected by spermine. However, there was an interaction of ARL 15896AR with the glycine regulatory site. In contrast to many uncompetitive NMDA antagonists, like MK-801, ARL 15896AR exhibited rapid kinetics. This property may result in a large margin of safety while maintaining the efficacy associated with use-dependent NMDA antagonists, making this compound an excellent candidate for clinical trials.