A Subtype of κ‐Opioid Receptor Mediates Inhibition of High‐Affinity GTPase Inherent in Gi1 in Guinea Pig Cerebellar Membranes

Abstract

The κ‐opioid receptor agonists including U‐50,488H and dynorphin A (1–17) in ranges of 0.1–100 nM inhibited the hydrolysis of GTP to GDP (P_i release) inherent in GTP‐binding proteins (G proteins) in guinea pig cerebellar membranes. U‐50,488H inhibited only high‐affinity GTPase activity, not low‐affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 µM. The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 µg/ml but not with preactivated cholera toxin (30 µg/ml). Similar blockade of κ‐receptor‐mediated inhibition was also observed when membranes were pretreated with a low concentration (8 µM) of N‐ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP‐ribosylated by PTX; but this treatment caused no significant change in κ‐agonist binding. When purified G_i1, but not G_o, was reconstituted into membranes pretreated with NEM, the κ‐receptor‐mediated inhibition was recovered. These findings suggest that a subtype of κ‐opioid receptor is coupled to inhibition of intrinsic activity of G_i1.