A natural processing product of rat diazepam binding inhibitor, triakontatetraneuropeptide (diazepam binding inhibitor 17-50) contains an alpha-helix, which allows discrimination between benzodiazepine binding site subtypes.
Synthetic peptides related to triakontatetraneuropeptide (TTN) [17TQPTDEEMLFIYSHFKQATVGDVNTDRPGLLDLK50; diazepam binding inhibitor (DBI) 17-50], a natural brain processing product of rat DBI, were analyzed for their physicochemical and ligand-receptor interaction characteristics. The ability of TTN and TTN-related fragments to displace [3H]flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5a] [1,4]-benzodiazepine-3-carboxylate) or [3H]Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1, 4-benzodiazepine-2-one] from their respective benzodiazepine (BZ) binding site subtypes was tested in intact cerebellar culture neurons or in homogenates of cultured astrocytes. These studies indicate that the C-terminal region of TTN, which is also present in DBI 22-50, eicosapentaneuropeptide (DBI 26-50), and octadecaneuropeptide (ODN) (DBI 33-50), but not in DBI 19-41, is essential for interaction with the BZ recognition sites. When the C-terminal lysine of ODN is blocked with an NH2 group, the ability of ODN to interact with the binding of [3H]flumazenil is lost. A comparison analysis of the binding data with the secondary structure characteristics of the peptides demonstrated that TTN (DBI 17-50) and DBI 22-50, which have hydrophobic portions and marked tendencies to produce alpha-helicity, specifically displace (apparent Ki, 5-6 microM) [3H] Ro 5-4864 from astroglial cell binding sites. Peptides (ODN, eicosapentaneuropeptide, OND-NH2) with very low tendencies to form alpha-helices and with virtually no hydrophobic structure were not able to displace Ro 5-4864 at concentrations of up to 100 microM. In contrast, ODN was a good displacer of [3H]flumazenil from intact neurons, with an apparent IC50 of 5 microM. These data suggest that the alpha-helical portion of TTN may be important for BZ receptor recognition and BZ receptor subtype discrimination.
Berkovich
A
A
Fidia-Georgetown Institute for the Neurosciences, Washington, DC 20007.
McPhie
P
P
Campagnone
M
M
Guidotti
A
A
Hensley
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001570:Benzodiazepinones / Q000378:metabolism; D001667:Binding, Competitive; D002942:Circular Dichroism; D005442:Flumazenil / Q000378:metabolism; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001570:Benzodiazepinones; D009479:Neuropeptides; D010446:Peptide Fragments; D011963:Receptors, GABA-A; C060941:triakontatetraneuropeptide; C028513:4'-chlorodiazepam; D005442:Flumazenil