ELSEVIER Neuroscience Letters 173 (19943 143-146 [[llIIl Dopamine and 7'-aminobutyric acid transporters: differential regulation by agents that promote phosphorylation Ye Tian, Gregory Kapatos, James G. Granneman, Michael J. Bannon* Cellular and ClinicalNeurobiology Program, Department of Psychiatry, and Department of Pharmacology, WayneState University, Detroil, :VII48201, USA Received 18 february 1994; Revised version received 7 April 1994; Accepted 7 April 1994 Abstract Treatment of striatal synaptosomes with the protein phosphatase inhibitor okadaic acid significantly decreased y-aminobutyric acid (GABA) uptake, indicating that the GABA transporter may be regulated by phosphorylation. Forskolin and 8-bromoadenosine- 3,5-cyclic monophosphate (8-br-cAMP) inhibited GABA uptake to the same extent as okadaic acid, suggestingthe involvement of protein kinase A in GABA transporter regulation. In contrast, the same treatments did not alter dopamine (DA) uptake into striatal synaptosomal preparations. The results suggest that the structurally related GABA and DA transporters may be subject to different post-translational regulation. Key words: Dopamine; ~,-Aminobutyric acid; Transporter; Uptake; Striatum; Phosphorylation The major mechanism for terminating the action of many released neurotransmitters is reuptake into either presynaptic neurons or nearby glial cells via specific and high-affinity transporters [for a review, see 1], The dopamine (DA) transporter (DAT) has been studied ex- tensively because of its involvement in the actions of drugs of abuse (e.g., cocaine and amphetamine) and its possible role in the etiology of Parkinson's disease [2,8], whereas the y-aminobutyric acid (GABA) transporter (GAT) has been suggested as a potential target for anti- convulsant and anxiolytic agents [13]. Based on their deduced protein sequence and ion-dependency, the DAT and GAT have been grouped into the same gene family [1]. Both the DAT and GAT contain putative consensus sites for protein phosphorylation [5], suggesting that their activity may be regulated by phosphorylation. Nev- ertheless, signal transduction mechanisms potentially regulating these transporters remain largely unexplored. * Corresponding author. Wayne State University, Department of Psy- chiatry, 2309 Scott Hall, 540 E Canfield, Detroit, M148201, USA. Fax: (1) (313) 993-4269. 0304-3940194l$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00298-0 In this study, various agents known to alter protein phos- phorylation were examined for effects on DAT and GAT activity in striatal synaptosomes. Male Sprague-Dawley (225 300 g) rats were obtained from Hilltop Laboratories (Portage, MI) and used in strict accordance with the NIH 'Guide to the Care and Use of Laboratory Animals'. DA HC1, GABA. cocaine HC1. aminooxyacetic acid HC1 (AOAA), tbrskolin, phorbol- 12,13-didecanoate. 8-bromoadenosine-3.5-cy- clic monophosphate (8-Br-cAMP), and pargyline were purchased from Sigma Chemical Co. (St. Louis. MOI. 3,4-[Ring-2,5,6-3H]DA and [2,3- 3H(N)]GABA were pur- chased from DuPont NEN (Boston. MA 1. Okadaic acid and nipecotic acid were obtained from Calbiochem t La Jolla. CA) and RBI (Natick. MAI, respectively Rats were killed by decapitation and a crude synap- tosomal fraction was prepared using a modified method of Gray and Whittaker [6]. Briefly, dissected striata were homogenized in 20 volumes of ice cold 0.32 M sucrose with 5 mM HEPES. ] mM EDTA (final pH 7.4), using a glass homogenizer and a teflon pestle (10 passes, 800 rpm). The homogenate was centrifuged at 1000 × g at 4°C for 10 min. The supernatant fluid (S1) was recentri- fuged at 15.000 × g at 4°C for 20 min. The resulting pellet