THE DOPAMINE TRANSPORTER AND CYTOCHROME-P450IID1 (DEBRISOQUINE 4-HYDROXYLASE) IN BRAIN - RESOLUTION AND IDENTIFICATION OF 2 DISTINCT [H-3] GBR-12935 BINDING-PROTEINS

Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P450IID1, 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 ∼ 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 (∼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 P450IID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 > budipine > α-lobeline > quinidine > α flupenthixol > SKF-525A > sparteine > 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 P450IID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P450IID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P450IID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P450IID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki ∼ 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 P450IID1, and that this radioligand may be a useful probe of P450IID1 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 P450IID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target Cells. © 1990.