Reserpine binding to chromaffin granules suggests the existence of two conformations of the monoamine transporter

The binding of 3H]reserpine (3H]RES) to purified bovine chromaffin granule membranes has been studied at low membrane concentration. Saturation isotherms indicated a dissociation equilibrium constant KD of 30 pM and a density of binding sites of 8 pmol/mg of protein at 30 degrees C. The association rate constant was 4.0 X 10(5) M-1 s-1, and the calculated dissociation rate constant was 1.2 X 10(-5) s-1, corresponding to a half-lifetime of about 16 h. Although this dissociation was too low to be measured directly, 3H]RES binding was indeed reversible since it was lost after addition of the detergent Triton X-100. Dihydrotetrabenazine (TBZOH) inhibited 3H]RES binding in a time-dependent manner, EC50 varying from 37 nM after a 1-h incubation to 600 nM after 16 h. On the contrary, 3H]RES binding inhibition by the substrate noradrenaline was time independent. It is proposed that the transporter exists in two different conformations which bind exclusively either tetrabenazine (TBZ) or RES and which are in equilibrium. The effects of detergents were consistent with this two-conformation model. The transporter solubilized by cholate bound 3H]TBZOH, but not 3H]RES. On the other hand, addition of cholate to membrane-bound 3H]RES solubilized the membrane without releasing the ligand from its binding site. It is proposed that the TBZ-binding conformation is obtained by solubilization with cholate and that RES stabilizes the RES-binding conformation, allowing its solubilization by this detergent.