Cationic Modulation of Human Dopamine Transporter: Dopamine Uptake and Inhibition of Uptake1
- 1Department of Pharmacology, Nanjing Medical University, Nanjing, Peoples Republic of China (N.C.); and 2Department of Chemistry, Emory University, Atlanta, Georgia (N.C., C.G.T., J.B.J.)
Abstract
Effects of cations on dopamine (DA) uptake into cells expressing the human dopamine transporter and on inhibition of DA uptake by various substrates and inhibitors were investigated by using rotating disk electrode voltammetry. The Na+ dependence of DA uptake varied with Na+ substitutes, hyperbolic with Li+, almost linear at 1 μM DA but hyperbolic at 8 μM DA with choline, and sigmoidal with K+. With Na+substituted by Li+, K[DA]decreased and Vapp remained constant with increasing [Na+], whereasK[Na+] decreased andVapp increased with increasing [DA], suggesting an ordered sequence with Na+binding before DA. Similar trends for the Na+-DA interactions were observed in the presence of cocaine. Cocaine inhibited DA uptake solely by increasing K[DA], with itsKi not significantly different at 55 and 155 mM [Na+], whereas it inhibited Na+stimulation by reducing Vapp more thanK[Na+] at 1 μM DA, andVapp only and less potently at 8 μM DA. Thus, cocaine may compete with DA, not with Na+, for the transporter, and might not follow a strictly ordered reaction with Na+. With Na+ substituted by K+, K[DA] orK[Na+] became insensitive to Na+ or DA. K+ impaired the DA uptake mainly by reducing Vapp, but affected cocaine inhibition by elevating Ki. Despite their different patterns for inhibiting DA uptake, nontransportable inhibitors cocaine, methylphenidate, mazindol, and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazine (GBR12909) showed similarly modest Na+ dependence in theirKi values. In contrast, substrates DA,m-tyramine, and amphetamine displayed a similarly stronger Na+ requirement for their apparent affinities.
Footnotes
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Send reprint requests to: Dr. Nianhang Chen, Department of Chemistry, 1515 Pierce Dr., Emory University, Atlanta, GA 30322-2210. E-mail: nchen{at}emory.edu
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↵1 This research was supported by National Institute on Drug Abuse (NIDA) Grant R03 DA10896. J.B.J. is a recipient of a NIDA Research Scientist Award (K02 DA00179).
- Abbreviations:
- DA
- dopamine
- DAT
- dopamine transporter
- hDAT
- human DAT
- RDE
- rotating disk electrode
- K[DA]
- concentration for DA to produce half-maximal initial rate of DA uptake at a given concentration of Na+
- KDA
- dissociation constant of DA at saturating concentrations of Na+
- K[Na+], concentration for Na+to produce half-maximal initial rate of DA uptake at a given concentration of DA
- KNa+, dissociation constant of Na+ at 0 concentration of DA
- Vapp
- apparent maximal initial rate of DA uptake at a given concentration of DA and saturating concentrations of Na+ or at saturating concentrations of DA and a given concentration of Na+
- Vmax
- maximal initial rate of DA uptake at saturating concentrations of DA and Na+
- GBR12909
- 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenyl-2-propyl)piperazine
- GBR12783
- 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine
- RTI-121
- 3β-(4-iodophenyl)tropane-2β-carboxylic acid isopropyl ester
- WIN 35,428
- 2β-carbomethoxy-3β-(4-fluorophenyl)tropane
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- Received January 26, 1999.
- Accepted April 2, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
