Activation of protein kinase C inhibits uptake, currents and binding associated with the human dopamine transporter expressed in Xenopus oocytes

• Published in: The Journal of pharmacology and experimental therapeutics 1997-09, Vol.282 (3), p.1358-1365
• Main Authors: Zhu, S J, Kavanaugh, M P, Sonders, M S, Amara, S G, Zahniser, N R
• Format: Article
• Language: English
• Subjects: Alanine - metabolism; Animals; Carrier Proteins - metabolism; Cyclic AMP-Dependent Protein Kinases - physiology; Dopamine - metabolism; Dopamine Plasma Membrane Transport Proteins; Humans; Mazindol - metabolism; Membrane Glycoproteins; Membrane Transport Proteins; Nerve Tissue Proteins; Oocytes - metabolism; Protein Kinase C - physiology; Recombinant Proteins - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Xenopus laevis
• ISSN: 0022-3565
• DOI: 10.1016/S0022-3565(24)36962-9

Activation of protein kinase C (PKC) regulates the activity of a number of neurotransmitter transporters. When Xenopus oocytes expressing the cloned human dopamine transporter (hDAT) were pretreated with bath-applied phorbol 12-myristate 13-acetate (PMA), a PKC activator, [3H]DA uptake decreased irreversibly in a time- and dose-dependent manner (IC50 = 22 nM; maximal inhibition = 63-85%). The inhibition appeared to be PKC-specific because incubation with the inactive form of phorbol ester 4alpha-phorbol-12,13-didecanoate (400 nM) did not change the uptake activity and PMA (100 nM) inhibition could be partially blocked by the selective PKC inhibitor bisindolylmaleimide I (1 microM). Saturation studies of [3H]DA uptake showed that PMA-induced inhibition was due to a decrease in V(max) with no change in K(T). Similar to uptake, PMA pretreatment inhibited both the hDAT transport-associated and substrate-independent leak currents. PMA also decreased membrane capacitance (C(m)) by 40%, selectively in hDAT-expressing oocytes. In addition, PMA pretreatment resulted in a 77% decrease in B(max) of [3H]mazindol binding to intact oocytes. In contrast, binding to whole homogenates of PMA-pretreated oocytes was not significantly altered. These results suggest that PMA regulates hDAT expressed in Xenopus oocytes by altering cell surface trafficking of hDAT.