Dissociation of guanosine 5'-[gamma-thio]triphosphate from guanine-nucleotide-binding regulatory proteins in native cardiac membranes. Regulation by nucleotides and muscarinic acetylcholine receptors.

Binding of the poorly hydrolyzable GTP analog, guanosine 5'-gamma-thio]triphosphate (GTPS]), to purified guanine-nucleotide-binding regulatory proteins (G proteins) has been shown to be nonreversible in the presence of millimolar concentrations of Mg2+. In porcine atrial membranes, binding of 35S]GTPS] to G proteins was stable in the presence of 1 mM Mg2+. However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTPS] greater than GTP greater than or equal to guanosine 5'-beta,gamma-imino]triphosphate greater than GDP greater than or equal to guanosine 5'-beta-thio]diphosphate greater than GMP, markedly enhanced the observed dissociation, with 20-30% of bound 35S]GTPS] being released by unlabelled guanine nucleotide within 20 min at 25 degrees C. Most interestingly, dissociation of 35S]GTPS] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetylcholine receptors. Carbachol-stimulated release of 35S]GTPS] was strictly dependent on the presence of Mg2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing 35S]GTPS] from the membranes by themselves, the guanine nucleoside triphosphates and diphosphates studied, at maximally effective concentrations, promoted the carbachol-induced dissociation to the same extent, while GMP and ATP were ineffective. GTPS]-binding-saturation experiments indicated that one agonist-activated muscarinic acetylcholine receptor can cause release of bound GTPS] from three to four G proteins. The data presented indicate that binding of GTPS] to G proteins in intact membranes, in contrast to purified G proteins, is reversible, and that agonist-activated receptors can even, either directly or indirectly, interact with GTPS]-bound G proteins, resulting in release of bound guanine nucleoside triphosphate.