Phorbol esters alter functions of the expressed dopamine transporter

Abstract

Recent elucidation of the amino acid sequences of the neurotransmitter transporters reveals several consensus sequences for phosphorylation by kinases including protein kinase C. Protein kinase C activation did modulate the function of the rat dopamine transporter expressed in COS cells. Cell treatment with the protein kinase C activator 12-myristate 13-acetate (PMA) reduced the affinity of binding of the radiolabeled cocaine analog [ 3H](-)-2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35,428) without affecting its B max. The uptake of [ 3H]dopamine was reduced by treatment with PMA in a staurosporine-sensitive manner. Kinetic analysis revealed that the inhibitory effect of PMA on [ 3H]dopamine uptake was due to reduced uptake velocity and a small reduction of affinity for Na +, without changed affinity for dopamine. 1-Oleoyl-2-acetyl- sn-glycerol (OAG) mimicked these actions of PMA. These results demonstrate that activation of protein kinase C alters dopamine transporter functions in both ligand recognition and substrate translocation. These phosphorylation phenomena in vitro suggest the possibility that phosphorylation could modulate the activity of this important dopaminergic synaptic regulator under physiological conditions.