ENDOGENOUS [H-3] FLUNITRAZEPAM BINDING IN HUMAN EMBRYONIC KIDNEY-CELL LINE-293

Specific endogenous [H-3]flunitrazepam binding sites were identified and characterized in membranes from the human embryonic kidney (HFK) cell line 293. A large part of these binding sites exhibited an intermediate affinity for [H-3]flunitrazepam and a mu M affinity for diazepam, clonazepam, 1-(2-chlorophenyl)-N-methyl-N-(1 -methylpropyl)-3-isoquinolinecarboxamide (PK 11195) or 4'-chlorodiazepam (Ro 5-4864). These sites, thus, resembled neither gamma-aminobutyric acid(A) (GABA(A)) receptor associated nor 'peripheral' benzodiazepine binding sites. A small part of the binding sites labeled by [H-3]flunitrazepam seemed to belong to 'peripheral' benzodiazepine binding sites exhibiting a nM affinity for PK 11195, and another small part of the binding sites seemed to exhibit a high affinity for flunitrazepam and PK 11195. Although small amounts of mRNA for (alpha(1)-, beta(3)- and gamma(2)-subunits of GABA(A) receptors could be identified in HEK 293 cells, neither the actual expression of GABA(A) receptors in these cells nor a coassembly of endogenous subunits with transfected GABA(A) receptor subunits could be demonstrated by binding studies.