The dopamine transporter and cytochrome P45OIID1 (debrisoquine 4-hydroxylase) in brain: resolution and identification of two distinct [3H]GBR-12935 binding proteins

Arch Biochem Biophys. 1990 Feb 1;276(2):424-32. doi: 10.1016/0003-9861(90)90741-g.


Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P45OIID1, were solubilized in digitonin from canine striatal membranes, and were resolved following wheat germ agglutinin (WGA)-lectin column chromatography. Protein adsorbed to and specifically eluted from WGA-lectin with N-acetylglucosamine displayed saturable, high affinity (KD approximately 3 nM), and sodium-dependent binding of [3H]GBR-12935, which was inhibited in a concentration-dependent and stereoselective manner by dopamine uptake blockers and substrates with a pharmacological profile indicative of the dopamine uptake site. Protein not adsorbed to WGA-lectin also bound [3H]-GBR-12935 with high affinity (approximately 7 nM), in a sodium-independent manner, and was insensitive to classical dopamine uptake blockers and substrates such as mazindol or dopamine, corresponding to the so-called "piperazine acceptor" site seen in native membranes. [3H]GBR-12935 binding to this latter protein was, however, inhibited by various compounds with a pharmacological profile indicative of a form of cytochrome P450 designated P45OIID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 greater than budipine greater than alpha-lobeline greater than quinidine greater than alpha flupenthixol greater than SKF-525A greater than sparteine greater than quinine. Ki values obtained for inhibition of [3H]-GBR-12935 binding to neuronal WGA passthrough fractions by these drugs correlate well with their respective Ki values for liver P45OIID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P45OIID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P45OIID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P45OIID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki approximately 100 nM) of sparteine monooxygenase activity in human liver microsomes. These data clearly indicate that [3H]GBR-12935 and its analogs display similar affinities for both the dopamine transporter and neuronal P45OIID1, and that this radioligand may be a useful probe of P45OIID1 activity in brain and liver. The exact molecular and functional association (if any) between these two distinct binding protein populations remains to be established; however, it is tempting to speculate that P45OIID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding, Competitive
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism*
  • Cattle
  • Cell Membrane / metabolism
  • Chromatography, Affinity
  • Corpus Striatum / metabolism*
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 Enzyme System / isolation & purification
  • Cytochrome P-450 Enzyme System / metabolism*
  • Dogs
  • Dopamine / metabolism*
  • Dopamine Plasma Membrane Transport Proteins
  • Kinetics
  • Ligands
  • Liver / metabolism*
  • Membrane Glycoproteins*
  • Membrane Transport Proteins*
  • Microsomes, Liver / enzymology
  • Mixed Function Oxygenases / isolation & purification
  • Mixed Function Oxygenases / metabolism*
  • Nerve Tissue Proteins*
  • Piperazines / metabolism*


  • Carrier Proteins
  • Dopamine Plasma Membrane Transport Proteins
  • Ligands
  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Piperazines
  • Cytochrome P-450 Enzyme System
  • 1-(2 (diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine
  • Mixed Function Oxygenases
  • Cytochrome P-450 CYP2D6
  • Dopamine