Uptake of the neurotoxin, 4-methylphenylpyridinium, into chromaffin granules and synaptic vesicles: A proton gradient drives its uptake through monoamine transporter

Energy dependence for uptake of 4-methyphenyl-pyridinium (MPP⁺), a neurotoxin causing Parkinsonism-like symptoms, by adrenal chromaffin granule membrane vesicles and brain synaptic vesicles was studied. The compound was actively taken up by the chromaffin vesicles dependent on hydrolysis of ATP with a K_m value of 22 μM and maximum velocity of 2.9 nmol/min/mg protein. The uptake was sensitive to reserpine (1 μM) and bafilomycin (50 nM) (inhibitors of the vesicular monoamine transporter and vacuolar-type H⁺-ATPase, respectively) and substrates for monoamine transporters, but insensitive to imipramine (an inhibitor of the monoamine transporter present in the plasma membrane). The uptake was greatly reduced upon dissipation of the proton gradient by ammonium ion or nigericin with KCl, but stimulated 1.6-fold by valinomycin plus K⁺. Dissipation of the proton gradient also induced rapid efflux of MPP⁺ from the vesicles. The MPP⁺ (monoamine) transporter was solubilized from chromaffin vesicles and reconstituted into liposomes with purified bacterial F₀F₁-ATPase. MPP⁺ was taken up by the liposomes coupled with ATP hydrolysis by F₀F₁, and the uptake was sensitive to reserpine, dissipation of the proton gradient, and azide. Brain synaptic vesicles also accumulated MPP⁺, showing similar kinetics, inhibitor sensitivities, and energy coupling to those of chromaffin vesicles. Furthermore, MPP⁺ inhibited the uptake of dopamine without affecting the uptake of glutamate or γ-aminobutyrate. These results indicated that MPP⁺ was taken up through the reserpine-sensitive monoamine transporter into chromaffin vesicles and synaptic vesicles and that the energy for accumulation of MPP⁺ was supplied as a proton gradient (acidic inside) established by H⁺-ATPase.