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

Si Jia Zhu, Michael P. Kavanaugh, Mark S. Sonders, Susan G. Amara, Nancy R. Zahniser

Research output: Contribution to journalArticlepeer-review

Abstract

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 4α-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 bisindolyl-maleimide 1 (1 μM). 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.

Original languageEnglish
Pages (from-to)1358-1365
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume282
Issue number3
StatePublished - Sep 1997

Fingerprint

Dive into the research topics of 'Activation of protein kinase C inhibits uptake, currents and binding associated with the human dopamine transporter expressed in Xenopus oocytes'. Together they form a unique fingerprint.

Cite this