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

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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]CR)‐(+)‐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 μM Hg2+, 10 μM Cu2+, and 10 μM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 μM was noncompetitive in nature, whereas 3‐5 μM Cu2+ afforded mixed‐type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1‐1 mM). Cu2+ (2 μM) did not affect the affinity of m‐flupenthixol or clo‐zapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 μM), Ni2+ (30 μM), 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 μM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.