Proton: substrate stoichiometries during active transport of biogenic amines in chromaffin ghosts

The relationship between the electrochemical proton gradient (delta mu-H+) and the electrochemical gradient for biogenic amines (delta mu-A) was investigated in isolated chromaffin ghosts free of endogenous components and gradients. The addition of ATP to a ghost suspension resulted in the generation of a large proton concentration gradient (delta pH), acidic inside (measured by [14C]-methylamine distribution), and a large proton electrical gradient (delta psi), positive inside (measured by [14C]-thiocyanate distribution). In the presence of this large electrochemical proton gradient, the accumulation of [14C]5-hydroxytryptamine (serotonin) and other biogenic amines rapidly reached an apparent steady state level. Collapse of the proton gradients after steady state levels were achieved resulted in the efflux of the accumulated amines. Uptake in the presence of a delta psi alone produced an amine gradient equal to the magnitude of the delta psi, while in the presence of a delta pH alone biogenic amine distribution was equal to twice the magnitude of the delta pH. Using additions of ammonia or thiocyanate, it was possible to vary the magnitude of the electrochemical proton gradient over a wide range of values; the driving force for amine accumulation under these conditions was found to be equal to delta psi--2Z delta pH (where Z = 2.3 RT/F). The results, which provide unequivocal evidence for the primary role of the electrochemical proton gradient in the active transport of biogenic amines, are consistent with a model based on the chemiosmotic hypothesis, of an obligatory coupling of amine influx and proton efflux, mediated via a putative reserpine-sensitive translocator. Measurement of the stoichiometry of this coupling of H+ and amines permits conclusions concerning the molecular mechanism of amino accumulation.