Activation of protein kinase C inhibits uptake, currents and binding associated with the human dopamine transporter expressed in Xenopus oocytes.
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.
Zhu
S J
SJ
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262, USA.
Kavanaugh
M P
MP
Sonders
M S
MS
Amara
S G
SG
Zahniser
N R
NR
D000409:Alanine / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D006801:Humans; D008454:Mazindol / Q000378:metabolism; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014982:Xenopus laevis
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D008454:Mazindol; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000409:Alanine; D004298:Dopamine