European Journal of Pharmacology - M o h , cular Pharmacology Section, 2(18( 1991) 269-270
~.'~1991 Elsevier Science Publishers B.V. All rights reserved 0922-4106/91/$9,3.50
Histidine residue is crucial for the binding of lig~nds to the benzodiazepine site except Ro15-4513 P e t e r B i n k l e y a n d M a h a r a j K. T i c k u Department of Pharmacolo,~', The Unicersity of Teras Health Science Center at San Antonio, San Antonio, TX 78284-7764, U.S.A.
Received I October 1991. accepted 7 October 1991
O u r laboratory has previously demonstrated that modification of the histidine residue with diethyl pyrocarbonate (DEP) inhibited the binding of ligands to the benzodiazepine (BZ) receptor site of the oligomeric G A B A A receptor complex (Burch et al., 1983). This effect was specific, since the same treatment did not alter G A B A binding, or the allosteric interaction between various sites on the G A B A a receptor complex (Burch et al., 1983). In addition, since D E P pretreatment inhibited the binding of BZ ligands completely in both cerebral cortex (type I; a t subunit a b u n d a n t ) and hippocampus (Type II; az a n d / o r a 3 abundant), it indicated that all the BZ receptor sites had a crucial histidine residue. Furthermore, since the inactivation could be prevented by BZ ligands like flurazepam, and not by other allosteric ligands, we speculated an involvement of a critical histidine residue at the BZ binding site (Maksay and Ticku, 1984). Ro15-4513, an imidazobenzodiazepine, which is structurally related to the BZ receptor antagonist, Ro15-1788, is known to bind to the BZ receptor site. Additionally, Ro15-4513 is known to bind to a diazepam insensitive site (57,000 Da) in the cerebellum (Sieghart et al., 1987). Recently, it has been demonstrated that the diazepam insensitive binding site which is recognized by Ro15-4513 in the cerebellum can be expressed in cells transfected with a~,[327 ~ cDNAs ( L u d d e n et al., 1990). In this report we examined the possibility that the D E P pretreatment might distinguish between different binding sites for Ro15-4513 in cerebral cortex and cerebellum. Male Sprague-Dawley rats (150-250 g) were used in the present study. The mitochondrial plus microsomal (P2 + P3) fraction was prepared, as described previ-
Correspondence to: Dr. M.K. Ticku, Department of Pharraacology. The Universityof Texas Health Science Center at San Anto:,,io.7703 Floyd Curl Drive. San Antonio. TX 78284-7764. U.S.A. Tel. (512) 567-4225; Fax (512) 567-4226.
ously (Burch et al., 1983). The membranes were treated with D E P (0.5-5 raM) for 20 rain at 0 ° C at pH 6.0, as described previously (Burch et al., 1983). Control tissue was treated in an identical manner, but without DEP. For binding studies, aliquots of control and DEPpretreated m e m b r a n e suspension in 0.05 M Tris HCI (pH 7.4) containing 0.05 M KCI were incnbated with [3H]Ro15-4513 (1 r~M, 60 min), [3H]flunitrazepam (l nM, 90 min) or [3H]Ro15-1788 (! nM, 60 min), and assayed by a filtration method (Burch et al., 1983). Nonspecific binding was measured in the presence of 10 -5 M Ro15-1788. Preliminary studies confirmed our earlier observation that D E P pretreatment (0.5-5 mM) inhibited [3H]flunitrazepam binding in a concentration dependent m a n n e r (data not shown). The effect of D E P was due to the decrease in the /3m,~ value (Burch et al. 1983). Table 1 summarizes the effect of D E P pretreatment on the binding of [3H]flunitrazepam, [3H]Ro151788 and [3H]Ro15-4513 in cerebral cortex and cerebellum. DEP (5 mM) pretreatment while inhibiting the binding of both [3H]flunitrazepam (agonist) and [3H]Ro15-1788 (antagonist) completely did not alter the
TABLE 1 Effect of DEP (5 mYl) pretreatment on the binding of radioligands to the benzodiazepine bindirg site of the GABAA receptor complex. Non~pecific binding was determined in the presence of 10 ~ M Ro15-1788. Vaiues represent mean _+S.D. of 4 -6 experiments, each done in triplicate. Specific binding (fmol/mg protein) Cerebral c o r t e x Cerebellum Control DEPControl DEPpretrea~:ed pretreated Flunitrazepam 177_+18 10_+ 4 t06+_7 8+_ 4 ( - 95Q ) ( - 92q~) [3H]Ro15-1788 t10_+19 5+_ 2 59_+5 3_+ 3 ( - 95c~) ( - 95%) [~H]RoI5-4513 90,2_ 8 81+1(1 58+-4 69_+11
specific binding of [3H]RoI5-4513 in either cerebral cortex or cerebellum. Additional studies were carried out to determine if DEP pretreatment affected the binding of Ro15-4513 to the diazepam insensitive site in cerebellum and cortex. Diazepam-insensitive sites were defined as described elsewhere (Turner et at., 1991). In agreement with a published report (Turner et al., 1991), we detected diazepam insensitive binding sites in both cerebellum and cerebral cortex (data not shown). DEP pretreatment (0.5-5 mM) did not alter the specific binding of [3H]RoI5-4513 to diazepam insensitive binding sites in either cerebellum or cerebral cortex (data not shown). These results indicate that there is a crucial histidine residue which is involved in the binding of ligands (e.g. agonists, antagonists) to the BZ receptor sites. This histidine appear~ to be present at all the BZ binding sites, since DEP inhibits the binding completely in both at subunit abundant cerebral cortex and o~2, a 3 subunit abundant hippocampus (Burch et al., 1983). However, Ro15-4513 binding to either diazepam sensitive or diazepam insensitive sites in cerebellum or cerebral cortex does not appear to involve this crucial histidine. These results indicate that the amino acid residues involved in the binding of Ro154513 are different from other ligands that bind to the BZ site of the oligomeric G A B A A receptor,
Acknowledgements This work was supported in part by grants from NIH (NS 15339) and ADAHMA (AA0409(1I. The authors thank Mrs. Sadie Phillips for excellent secretarial assistance.
References Butch, T.P., R. Thyagarajan and M.K. Ticku, 1983, GrG~lp selective modification of the benzodiazepine ~,,-aminob~.'tyric acid receptor-ionophore complex reveals that low-affinity y-aminobutyric acid receptors stimulate benzodiazepine binding, Mol. Pharmacol. 23, 52. Luddens, lq., D.B. Pritchen, M. Kohler, I. Killisch, K. Ke.:nanen, H. Monyer, R Sprengel and P.H. Seeburg, 1990, Cerebellar GABA A receptor selective for a behavioral alcohol antagonist, Nature 346, 648. Maksay, G. and M.K. Ticku, 1984. Characterization of GABA-benzodiazepine receptor complex by protection against inactivation by group-specific reagents, J. Neurochem. 42, 1715. Sieghart. W., A. Eichinger, J.G. Richards and H. M6hler, 1987, Photoaffini~y labelling of benzodiazepine receptor protein with the partial inverse agonist [3H]RoI5-4513: a biochemical and autoradiographic study, J. Neurochem. 46, 46. Turner, D.M., D.W. Sapp and R.W. Olsen, 1991, The benzodiazepine/alcohol antagonist Ro15-45t3: binding to a GABA n receptor subtype that is insensitive to diazepam, J. Pharmaeol. Exp. Ther. 257, 1236.