[2-3H]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]-quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane ofpurified bovine chromaffin granules. The chromaffin granules of the adrenal medulla and the ghosts derived from their membranes accumulate catecholamines by a two-step ATP-dependent process (1-4): (i) inward translocation of protons by an electrogenic ATP-dependent H' pump (5-10), and (ii) a monoamine carrier driven by the H' electrochemical gradient (9,(11)(12)(13)(14)(15) and specifically blocked by reserpine or tetrabenazine (16). Whereas the H' pump has been functionally and structurally related to the mitochondrial ATPase complex (17,18), the catecholamine carrier has not yet been characterized. It is assumed to be a protein because the transport is temperature sensitive, shows some stereoselectivity (19), and is affected by the histidine-specific reagent diethylpyrocarbonate (20). The carrier has been solubilized and reconstituted in lipid vesicles (21,22), but this technique has not allowed purification. The carrier activity is difficult to assay because vesicles have to be energized to accumulate catecholamines, thus introducing possible artifacts.An alternate approach to the study of the carrier is the use ofbinding techniques. We have recently shown that, of several inhibitors (tetrabenazine, reserpine, haloperidol, and chlorpromazine), tetrabenazine has the most specific interaction with the carrier (23). In the present communication, we describe the binding of 2-[2-3H]hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3, 4,6,7-hexahydro-llb-H-benzo[a]quinolizine (dihydrotetrabenazine, [3H]TBZOH), a derivative of tetrabenazine to bovine chromaffin granule membrane. A preliminary account of some of these experiments has been published (24).[3H]Dihydrotetrabenazine MATERIALS AND METHODS TBZOH was obtained by reduction of tetrabenazine (Fluka) by NaBH4 in methanol (25).[3H]TBZOH (12 Ci/mmol; 1 Ci = 3.7 x 1010 Bq) was prepared as described (24). Its purity was periodically checked by TLC and, when necessary, the product was repurified (24). Stock solutions (80 ,AM) were made in 100 mM HCl and diluted in water.Bovine chromaffin granule membranes were prepared by osmotic lysis of granules isolated by centrifugation on a 1.6 M sucrose layer (26,27). Membranes were frozen in liquid nitrogen and were stored at -80°C. They were rapidly thawed at 370C, centrifuged at 100,000 x g for 15 min, and resuspended -in 20 mM Hepes KOH buffer containing 0.3 M sucrose.For [3H]TBZOH binding studies, membranes (5-15 ,ug of protein per ml) were incubated at 25°C with various concentrations of [3H]TBZOH in 0.3 M sucrose/20 mM KOH Hepes, pH 7.5. Bound ligand was measured by filtration on Millipore HAWP filters or by centrifugation at full speed in a Beckman Airfuge with cellulose propionate tubes. For filtration, 0.2-to 2.0-ml aliquots of the incubation mixture were diluted in 4 ml of ice-cold 0.3 M sucrose/10 mM KOH Hepes, pH 7.5, containing 125 ...

An adiabatic conformational analysis of serotonin (5-hydroxytryptamine, 5-HT) using quantum chemistry led to six stable conformers that can be either +gauche (Gp), -gauche (Gm), and anti (At) depending upon the value taken by ethylamine side chain and 5-hydroxyl group dihedral angles φ1, φ2, and φ4, respectively. Further vibrational frequency analysis of the GmGp, GmGm, and GmAt conformers with the 5-hydroxyl group in the anti position revealed an additional red-shifted N-H stretch mode band in GmGp and GmGm that is absent in GmAt. This band corresponds to the 5-HT side-chain N-H bond involved in an intramolecular nonbonded interaction with the 5-hydroxy indole ring. The influence of this nonbonded interaction on the electronic distribution was assessed by analysis of the spin-spin coupling constants of GmGp and GmGm that show a marked increase for C2-C3 and C8-C9 bonds in GmGm and GmGp, respectively, with a decrease of their double bond character and an increase of their length. The Atoms in Molecules (AIM), Natural Bond Orbital (NBO), and fluorescence and CD spectra (TDDFT method) analyses confirmed the existence in GmGp and GmGm of a through-space charge-transfer between the HOMO and the HOMO-1 π-orbital of the indole ring and the LUMO σ* N-H antibonding orbital of the ammonium group. The strength of the cation-π interaction was determined by calculating binding energies of the NH4(+)/5-hydroxyindole complexes extracted from stable conformers. The energy decomposition analysis indicated that cationic-π interactions in the GmGp and GmGm conformers are governed by the electrostatic term with significant contributions from polarization and charge transfer. The lower stability of the GmGm over the GmGp comes from a higher exchange repulsion and a weaker polarization contributions. Our results provide insight into the nature of intramolecular forces that influence the conformational properties of 5-HT.