Spontaneous association between opioid receptors and GTP-binding regulatory proteins in native membranes: specific regulation by antagonists and sodium ions.

High affinity GTPase in membranes from NG108-15 cells was differentially affected by opioid competitive antagonists; one type of antagonist [( N,N'-diallyl-Tyr1-Aib2,3]Leu-enkephalin) reduced the basal rate of GTP hydrolysis, whereas a second type (MR 2266) produced no changes. The inhibitory effect of the "active" antagonist was stereospecifically reversed by the "inactive" antagonist, indicating that it was receptor mediated. This suggests that part of basal GTPase activity in this system results from a spontaneous interaction between opioid receptors and GTP-binding proteins (G proteins) and that some antagonists exhibit negative intrinsic activity by hindering such an interaction. The inhibitory effect of the antagonist was minimal in the presence of Na+ and maximal when Na+ was replaced by K+ in the reaction. When the ratio [Na+]/[K+] was progressively increased at constant [Cl-], total GTPase activity (i.e., net difference between activity stimulated by agonist and that inhibited by antagonist) did not change, but the activity measured in the absence of ligand was selectively decreased. Thus, Na+ does not alter the total proportion of G proteins that can be activated by ligand-occupied receptors and instead regulates the interaction between receptor and G protein in the absence of ligand. Upon examination of several opioid agonist and antagonists, we found an inverse relation between the intrinsic activity (either negative or positive) of each ligand and the sensitivity to Na+ of the GTPase elicited upon occupation of the receptor by that ligand. Sodium-mediated and ligand-mediated regulations of GTPase had identical requirements for Mg2+ [( Mg2+]free greater than 10 microM), and were both abolished with a similar potency by pertussis toxin. There was no effect of Na+ on the basal rate of GTP hydrolysis of Gi/Go purified from bovine brain. However, addition of these proteins to membranes prepared from cells that had been previously exposed to pertussis toxin partially restored both receptor- and sodium-mediated regulations of GTPase in parallel and in a concentration-dependent fashion. We conclude that sodium ions play a key role in the mechanism underlying the spontaneous interaction between "empty" receptors and G proteins in intact membranes.