title: Effects of heavy metal cations and other sulfhydryl reagents on brain dopamine D1 receptors: evidence for involvement of a thiol group in the conformation of the active site.
abstract: To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.
mesh_terms: D000818:Animals; D001552:Benzazepines / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008670:Metals / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000187:drug effects* / Q000378:metabolism; D017447:Receptors, Dopamine D1; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
chemical_list: D001552:Benzazepines; D002413:Cations, Divalent; D008670:Metals; D011954:Receptors, Dopamine; D017447:Receptors, Dopamine D1; D013439:Sulfhydryl Reagents; D008274:Magnesium; D005033:Ethylmaleimide; D004229:Dithiothreitol