An electrophilic affinity ligand based on (+)-MK801 distinguishes PCP site 1 from PCP site 2.

The electrophilic affinity ligand, (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycl ohepten-5,10 - imine hydrochloride [(+)-MK801-NCS] was characterized for its ability to acrylate phencyclidine (PCP) and sigma binding sites in vivo. Initial studies, conducted with mouse brain membranes, characterized the binding sites labeled by [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP). The Kd values of [3H]TCP for PCP site 1 (MK801-sensitive) and PCP site 2 (MK801-insensitive) were 12 nM and 68 nM, with Bmax values of 1442 and 734 fmol/mg protein, respectively. Mice were sacrificed 18-24 hours following intracerebroventricular administration of the acylator. The administration of (+)-MK801-NCS increased [3H]TCP binding to site 2, but not to site 1. Although (+)-MK801-NCS decreased [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d; ccyclohepten-5,10-imine maleate ([3H](+)-MK801) binding to site 1, it had no effect on [3H]TCP binding to site 1. Viewed collectively with other published data, these data support the hypothesis that PCP sites 1 and 2 are distinct binding sites, and that [3H]TCP and [3H](+)-MK801 label different domains of the PCP binding site associated with the NMDA receptor.