Endogenous [ 3H]flunitrazepam binding in human embryonic kidney cell line 293

Abstract

Specific endogenous [ 3H]flunitrazepam binding sites were identified and characterized in membranes from the human embryonic kidney (HEK) cell line 293. A large part of these binding sites exhibited an intermediate affinity for [ 3H]flunitrazepam and a μM 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 γ-aminobutyric acid A (GABA A) receptor associated nor ‘peripheral’ benzodiazepine binding sites. A small part of the binding sites labeled by [ 3H]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 α 1-, β 3- and γ 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.