Regulation of Guanine Nucleotide Turnover on Gi/Goby Agonist-Stimulated and Spontaneously Active Muscarinic Receptors in Cardiac Membranes

Abstract

Muscarinic receptor regulation of guanine nucleotide turnover on Gi/Goproteins in ventricular sarcolemma was investigated. In the absence of a muscarinic receptor (MR) agonist, GTP bound to background sites with aKappvalue of 60 nM and aBmaxof 50 pmol/mg. The addition of the MR agonist, carbachol, further increased GTP binding by 50 pmol/mg to sites with the sameKappvalue of 60 nM. Pertussis toxin treatment reduced GTP binding to carbachol-regulated and background binding sites, thus identifying both sites as Gi/Go. The identity of the carbachol-regulated GTP binding sites was further confirmed by demonstrating that carbachol stimulated GTP binding and inhibited adenylyl cyclase with an EC50value of 200 nM. Background and carbachol-regulated guanine nucleotide binding sites bound GDP with aKappvalue of 150 nM. However, maximal background GDP binding was 50 pmol/mg, whereas maximal carbachol-regulated GDP binding was only 12–15 pmol/mg. In sarcolemma preloaded with [3H]GDP, carbachol-regulated [3H]GDP release was strictly dependent on the presence of guanine nucleotides. TheKappvalues for GTP and GDP to support carbachol-regulated [3H]GDP release were 60 nM and 150 nM, respectively. Guanosine 5′-O-(3-thiotriphosphate) (GDPβS) facilitated carbachol-regulated [3H]GDP release with aKappvalue of 2 μM. However, GTP was two times more efficacious than GDP or GDPβS in facilitating carbachol-regulated [3H]GDP release. Mn2+also stimulated [3H]GDP release from carbachol-regulated sites by a mechanism not requiring guanine nucleotides. These studies indicate that two pools of muscarinic receptors, carbachol regulated and spontaneously active, regulate guanine nucleotide turnover on pertussis toxin sensitive Gi/Go. These studies further suggest that guanine nucleotide binding provides the signal to stimulate GDP release from receptor activated Gi/Goproteins. A quaternary mechanism involving G-protein interactions may be necessary to promote guanine nucleotide exchange on Gi/Go.