Species differences in functions of dopamine transporter: paucity of MPP+ uptake and cocaine binding in bovine dopamine transporter

ELSEVIER

NeuroscienceLetters 214 (1996) 199-201

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Species differe,nces in functions of dopamine transporter: paucity of MPP + uptake and cocaine binding in bovine dopamine transporter Sang-Hun Lee, Jinseol Rhee, Jai-Kyung Koh, Yong-Sung Lee* Department of Biochemistry, College of Medicine, Hanyang University, # 17 Haengdang-dong, Sungdong-Ku, Seoul, 133-791, South Korea

Received 18 June 1996; revisedversionreceived 16 July 1996;accepted 16 July 1996

Abstract

Expression of cloned cDNA for human, rat and bovine dopamine transporter (DAT) in COS cells allows the comparison of the functional differences among the respective dopamine transporters. Human DAT showed the highest activities for dopamine uptake, MPP ÷ uptake and cocaine binding, indicating that humans are more vulnerable to 1-methyl-4-phenylpyridinium (MPP ÷) toxicity and cocaine addiction. However, bovine DAT showed poor MPP ÷ uptake and cocaine binding, even though its dopamine uptake ability was quite avid. Here, we conclude that the paucity of MPP + uptake and cocaine binding is a unique characteristic in bovine DAT. Keywords: Dopamine transporter; Dopamine uptake; 1-methyl-4-phenylpyridinium uptake; Cocaine binding

Dopamine transporter (DAT) is a highly specialized membrane-spanning protein that aids in terminating dopaminergic neurotransmission by sodium-dependent reuptake of dopamine (DA) released into synaptic clefts [1-5]. Besides the nomaal physiologic role of dopamine transporting, DAT has adverse functions as a gate for dopaminergic neurotoxms such as 1-methyl-4-phenylpyridinium (MPP ÷) [6-8], and a specific cocaine receptor [3,9]. A comparison of the deduced amino acid sequences of the cloned DATs reveals a high level primary and putative secondary structure conservation. Nevertheless, the bovine DAT sequence is slightly different from those of rats and humans, postulating that some functions of bovine DAT would be dissimilar from the others. In this study, we present that cloned bovine DAT shows very little cocaine binding and MPP ÷ transporting activities. COS cells were transfected with each of bovine, human and rat DAT cDNA subcloned into the eukaryotic expression vector, pRc/CMV, by calcium-phosphate DNA precipitation [10], and weie allowed to express their cDNAs for 2 days, then assayed for their abilities to accumulate [3H]DA or [3H]MPP+ or the abilities for their membranes to bind the tritium l~tbeled cocaine analog 2B-carbo* Correspondingauthor. Tel.: +82 2 2908242; fax: +82 2 2946270; e-mail: [email protected]~tg.ac.kr

methoxy-3~-(4-fluorophenyl)tropane ([3H]CFT, also designated [3H]WIN35428). Cells (0.5 × 105 cells per well) were plated on 24-well plates in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, transfected with 0.3/~g of pRc/CMV carrying each DAT cDNAs which had been precipitated in 0.125 M CaC12, 1 x BES-buffered solution (BBS; 50 mM BES, 280 mM NaC1, 1.5 mM sodium phosphate (pH 6.95)). Cells were cultured for 2 days, then uptake and binding assays were performed in a modified Krebs-Ringer HEPES (KRH) buffer containing 25 mM HEPES (pH 7.4), 125 mM NaCI, 4.8 mM KC1, 1.2 mM KH2PO4, 1.3 mM CaC12, 1.2 mM MgS04, 5.6 mM glucose, 1 mM sodium ascorbate (for [3H]DA uptake, 10/~M pargyline was added). Cells were washed and preincubated in the modified KRH buffer for 10 min at 37°C, and then incubated with 50 nM [3H]DA (51 Ci/mmol; Amersham Co., England) or 10 nM [3H]MPP+ (83 Ci/mmol; DupontNew England Nuclear Co., Boston, MA, USA) for 10 min at 37°C. The uptake reactions were terminated by three washes with ice-cold buffer. Cells were solubilized in 0.5 M NaOH and radioactivity was assessed by liquid scintillation spectroscopy. The binding of [3H]CFT to DATs on intact cell membranes was determined by incubating cells with the same buffer containing 10 nM [3H]CFT (87 Ci/ mmol; Dupont-New England Nuclear Co., Boston, MA,

0304-3940/96/$12.00 © 1996 ElsevierScienceIreland Ltd. All rights reserved PH S0304-3940(96) 1291~3-X

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S.-H. Lee et al. / Neuroscience Letters 214 (1996) 199-201

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Fig. 1. Comparisons of DAT functions between species. Uptakes were assessed with 50 nM [3H]DA for 10 min (DA uptake assay) or 10 nM [3H]MPP+ for 90 min (MPP + uptake assay) at 37°C. CFT binding was determined by incubation with 10 nM [3H]CFF at 4°C for 120 min, as described in the text. Columns represent the means and vertical lines represent SEM.

USA) for 120 min at 4°C. Specific uptakes or binding were defined by subtracting the uptakes (or binding) of the cells transfected with intact pRc/CMV vector (not carrying neither DAT cDNAs), in the identical conditions, from the respective uptake and binding values of the human, rat and bovine transfection. Saturation analyses were performed with 50 nM [3H]DA and unlabeled DA (0.5-100 /xM) or with 5-300 nM [3H]CFT. Saturation analysis of [3H]MPP+ uptake could not be carried out because of cellular toxicity of MPP ÷ in high dose (higher than 10 /zM). COS cells transfected with pRc/CMV carrying bovine, rat, or human DAT displayed avid uptake of [3H]DA (0.44 + 0.13, 0.40 + 0.02, and 0.62 + 0.06 pmol]105 cells per 10 min, respectively), which were relatively similar with each other (Fig. 1). However, the abilities of cocaine binding and MPP + transporting were quite different between species. The binding ability of [3H]CFT of membranes in COS cells expressing human DAT (172.89 + 15.56 fmol/105 cells) was higher than other species. [3H]CFT bindings of membranes in cells expressing DATs of rat and bovine were 47% and 10% of that in human DAT, respectively (Fig. 1). Saturation analyses and kinetic parameters of DA uptakes and CFT bindings are shown in Fig. 2 and Table 1. Accumulation of [3H]MPP + into rat, or human DAT-expressing cells was also rapid and considerable. During 10 min of incubation, uptakes of [3H]MPP+ by rat or human DAT were increased sharply, and reach to a level of 43 and 67 fmol/105 cell, respectively. As the incubation period increased, the level of MPP + uptake leveled off after 10 min (Fig. 3). However, bovine DAT was nearly devoid of

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[3H]CFT (nM) Fig. 2. Saturation analyses of (A) [3H]DA uptake and (B) [3H]CFr binding of COS cells expressing each DAT cDNA. Cells were preincubated in a modified-KRH buffer at 37°C for 15 min. DA uptake was assessed by incubation with 50 nM [3H]DA and unlabeled DA (0.5-100 /~M) at 37°C for 10 min. CFT binding was assessed by incubation with 5-300 nM [3H]CFr for 120 min at 4°C. Data are the mean + SEM of triplicate determinations.

MPP + uptake. Accumulation of [3H]MPP ÷ by bovine DAT for 10 min was almost undetectable in given conditions, and only slightly increased with an increasing incubation time to 90 min but still did not exceed 4% of that found in human DAT (Figs. 1 and 3). These results of MPP + uptake are consistent with the in vivo study performed by Langston and Irwin [11]. They have reported that primates are much more susceptible to the parkinsoTable 1 Kinetic parameters of [3H]DA uptake and [3H]CFT binding in DATs DAT

[3H]DA uptake

[3H]CFT binding

Km (~M)

Vm~ (pmoF 105 cells)

Kd ( ~ )

Bmax ( ~ o ~ l0 s cells)

17.48 + 1.40 6.72 + 0.51 8.26 + 0.47

43.76 + 10.74 34.31 + 12.00 8.29 + 3.08

330.00 + 26.72 155.60 + 16.88 34.62 + 2.48

Human 1.72 + 0.43 Rat 1.59 + 0.38 Bovine 0.99 + 0.21

Values represent the mean + SEM of Iriplicate determinations.

S.-H. Lee et al. / Neuroscience Letters 214 (1996) 199-201

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nogenic effects ofMPP ÷ than other species, and our results imply that this susceptibility is particularly due to the high uptake of MPP ÷ by human DAT. In addition, the highest cocaine binding to human DAT can also implicate that humans are more susceptible to the effects and addiction of cocaine than other species. Considering that DA uptake of bovine DAT was eqluivalent to those of rat and human DATs in identical experimental conditions, only negligible quantities of MPP ÷ uptake and scarcity of cocaine-binding are significant characteristics of bovine DAT. Even though the DAT has been studied extensively because of its involvement in the action of cocaine addiction and its possible role in the etiology of Parkinson's disease, even the recognition/binding sites for DA, MPP + and cocaine remain unexplored. In this study, we showed that the paucity ofMPP ÷ uptake and cocaine binding is an intrinsic characteristic of bovine DAT. Kitayama et al., based on a mutation study on rat DAT [12], have suggested that the l lth hydrophobic transmembrane domain is crucial specifically for the ability of MPP ÷ uptake, implying that any differences between rat and bovine DAT sequence of the l lth domain can explain the paucity of MPP ÷ uptake of bovine DAT in our result. However, the amino acid sequence of 1 lth transmembrane domain in bovine DAT is almost identical to that of the

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other DATs. Only three out of 22 amino acid residues are different between bovine and rat DATs. Moreover, each three different residues can be substituted conservatively between species. Therefore, the deficiency of MPP + uptake in bovine DAT would not be explained by the suggestion by Kitayama et al. When the amino acid sequences of cloned DATs are compared, bovine DAT is relatively dissimilar with the others (in overall amino acids sequence, human DAT is 92% identical to the rat and 84% identical to the bovine DAT). Especially, the intracellular carboxyl end (amino acid position 577-693) of bovine DAT is 2.6 times longer than that of human or rat DAT (an additional 72 amino acids exists on carboxyl end of bovine DAT). These differences in bovine DAT can contribute to the paucity of MPP + uptake and cocaine binding. The analyses of unique sequences in bovine DAT is expected to shed light into the discovery of the functional sites and action mechanisms for DAT. We thank Dr. Marc Caron and Dr. Tedd Usdin for providing us DAT cDNA clone. [1] Iversen, L.L., Role of transmitter uptake mechanisms in synaptic neurotransmission, Br. J. Pharmacol., 41 (1971) 571-591. [2] Horn, A.S., Dopamine uptake: a review of progress in the last decade, Prog. Neurobiol., 34 (1990) 387-400. [3] Shimada, S., Kitayama, S., Lin, C.L., Patel, A., Nanthakumar, E., Gregor, P., Kuhar, M. and Uhl, G., Cloning and expression of a cocaine-sensitive dopamine transporter complementary DNA, Science, 254 (1991) 576-578. [4] Uhl, G.R., Neurotransmitter transporters (plus) promissing new gene family, Trends Neurosci., 15 (1992) 265-268. [5] Uhl, G.R. and Hartig, P.R., Transporter explosion: update on uptake, Trends Pharmacol. Sci., 13 (1992) 421-425. [6] Kitayama, S., Shimada, S. and Uhl, G.R., Parkinsonism-inducing neurotoxin MPP÷: uptake and toxicity in nonneuronal COS cells expressing dopamine transporter cDNA, Ann. Neurol., 32 (1992) 109-111. [7] Snyder, S.H. and D'Amato, R.J., MPTP: a neurotoxin relevent to the pathophysiology of Parkinson's disease, Neurology, 36 (1986) 250-258. [8] Uhl, G.R., Parkinson's disease: neurotransmitter and neurotoxin receptors and their genes, Eur. Neurol., 30 (1990) 21-30. [9] Usdin, T.B., Mezey, E., Chert, C., Brownstein, M.J. and Hoffman, B.J., Cloning of the cocaine-sensitive bovine dopamine transporter, Proc. Natl. Acad. Sci. USA, 88 (1991) 11168-11171. [10] Ausubei, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (Eds.), Short Protocols in Molecular Biology, Greene Publishing Associates and Wiley, New York, 1992, 9.5 p. [11] Langston, J.W. and Irwin, I., MPTP; current concepts and controversies, Clin. Neuropharmacol., 9 (1986) 485-507. [12] Kitayama, S., Wang, J.-B. and Uhl, G.R., Dopamine transporter utants selectively enhance MPP transport, Synapse, 15 (1993) 58-62.