Activation of Protein Kinase C Inhibits Uptake, Currents and Binding Associated with the Human Dopamine Transporter Expressed inXenopus Oocytes1
- 1Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado (S.J.Z., N.R.Z.) and 2Vollum Institute, Oregon Health Sciences University, Portland, Oregon (M.P.K., M.S.S., S.G.A.)
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 bisindolylmaleimide I (1 μM). Saturation studies of [3H]DA uptake showed that PMA-induced inhibition was due to a decrease in Vmax with no change in KT. Similar to uptake, PMA pretreatment inhibited both the hDAT transport-associated and substrate-independent leak currents. PMA also decreased membrane capacitance (Cm) by 40%, selectively in hDAT-expressing oocytes. In addition, PMA pretreatment resulted in a 77% decrease in Bmax 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.
Footnotes
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Send reprint requests to: Dr. Nancy R. Zahniser, Department of Pharmacology C236, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262.
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↵1 This work was supported by National Institutes of Health grant DA04216, postdoctoral fellowship DA50706 to S.J.Z. and career development award DA05706 to N.R.Z.
- Abbreviations:
- BIM
- bisindolylmaleimide I
- Cm,membrane capacitance
- DA, dopamine
- DAT
- dopamine transporter
- DMSO
- dimethyl sulfoxide
- FRB
- frog Ringer’s buffer
- GABA
- γ-aminobutyric acid
- hDAT
- human dopamine transporter
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- I-V
- current-voltage
- 4αPDD
- 4α-phorbol-12,13-didecanoate
- PKA
- cAMP-dependent protein kinase
- PKC
- protein kinase C
- PMA
- phorbol 12-myristate 13-acetate
- rDAT
- rat dopamine transporter
- SDS
- sodium dodecyl sulfate
- SERT
- serotonin transporter
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- Received January 29, 1997.
- Accepted May 27, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
