Dissociation of guanosine 5′‐[γ‐thio]triphosphate from guanine‐nucleotide‐binding regulatory proteins in native cardiac membranes

Binding of the poorly hydrolyzable GTP analog, guanosine 5′‐[γ‐thio]triphosphate (GTP[S]), to purified guanine‐nucleotide‐binding regulatory proteins (G proteins) has been shown to be non‐reversible in the presence of millimolar concentrations of Mg 2+ . In porcine atrial membranes, binding of [ 35 S]GTP[S] to G proteins was stable in the presence of 1 mM Mg 2+ . However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTP[S] > GTP ≥ guanosine 5′‐[β,γ‐imino]triphosphate > GDP ≥ guanosine 5′‐[β‐thio]diphosphate > GMP, markedly enhanced the observed dissociation, with 20–30% of bound [ 35 S]GTP[S] being released by unlabelled guanine nucleotide within 20 min at 25°C. Most interestingly, dissociation of [ 35 S]GTP[S] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetycholine receptors. Carbachol‐stimulated release of [ 35 S]GTP[S] was strictly dependent on the presence of Mg 2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing [ 35 S]GTP[S] 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. GTP[S]‐binding‐saturation experiments indicated that one agonist‐activated muscarinic acetylcholine receptor can cause release of bound GTP[S] from three to four G proteins. The data presented indicate that binding of GTP[S] 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 GTP[S]‐bound G proteins, resulting in release of bound guanine nucleoside triphosphate.