Reserpine binding to bovine chromaffin granule membranes. Characterization and comparison with dihydrotetrabenazine binding.

[3H]Reserpine bound reversibly in vitro to chromaffin granule membranes. Binding was temperature-dependent and slow, and had biphasic kinetics. The addition of ATP accelerated the kinetics, which became monophasic and comparable to those of [3H] dihydrotetrabenazine, without affecting the binding equilibrium constants. The ATP effect was related to H+ -electrochemical gradient generation by the granule membrane H+ pump. Binding of reserpine to chromaffin granule membranes occurred on two classes of sites: R1, Bmax = 7 pmoles/mg of protein and KD = 0.7 nM, and R2, Bmax = 60 pmoles/mg of protein and KD = 25 nM. Sites R2 were considered to be equivalent to [3H] dihydrotetrabenazine binding sites, as the densities of the R2 and the [3H] dihydrotetrabenazine binding sites were similar and because tetrabenazine displaced reserpine from R2 sites. Sites R1 were tetrabenazine-resistant; they were involved in monoamine uptake, since their KD values were similar to the KI values of reserpine for noradrenaline uptake. Sites R1 were less abundant than sites R2 on chromaffin granule membranes, but they were present at the same concentration in intact chromaffin granules. We propose that the monoamine carrier exists in two forms: (a) an active form bearing both high- and low-affinity sites for reserpine and (b) an inactive form with only the low-affinity R2 sites.