Comparison of agonist occupancy of M2 muscarinic receptor‐G protein complexes in myocardial homogenates with functional estimates of active receptor‐state affinity in isolated atria

Abstract

We have recently developed methods for estimating the active (Kact) and inactive (Kinact) receptor‐state affinity constants of ligands in functional studies on G protein‐coupled receptors. Our approach is valid for in vitro responses measured downstream from receptor activation, such as second messenger signaling in cell lines and responses in isolated tissues. The data required for our analysis include agonist‐mediated responses measured in the absence and presence of reduced receptor expression and modulation with an allosteric agonist (J Pharmacol Toxicol Methods 69:253–279, 2014). We also described an analogous technique that employs a constitutively active receptor mutant (J Pharmacol Toxicol Methods 83:94–106, 2017) instead of an allosteric agonist. In this study, we sought to validate our functional determination of Kact by using an independent biophysical method for estimating Kact. It is well known that muscarinic agonists exhibit high affinity for a subset of M2 muscarinic receptors in myocardial homogenates as measured in competitive binding experiments with [3H]N‐methylscopolamine. This subset of muscarinic binding sites is thought to represent a receptor‐Gi/o protein complex. We measured agonist binding to this site directly using the agonist radioligand, [3H]oxotremorine‐M, and completed agonist/[3H]oxotremorine‐M competitive binding experiments to estimate ligand affinity for this site in the guinea pig myocardium. We estimated a log affinity constant of approximately 8.2 for oxotremorine‐M in competitive binding experiments with [3H]oxotremorine‐M in the presence of 140 mM Na+. The allosteric agonist, LY211,9620 caused about a twofold increase in the affinity of oxotremorine‐for this site. In the presence of low sodium, LY211,9620 had little effect on oxotremorine‐M affinity, suggesting that in presence of both LY211,9620 and low sodium, the observed affinity of oxotremorine‐M is nearly the same as the active state affinity. Under these conditions, the log affinity constant of oxotremorine‐M was approximately 8.8. This value was similar to that estimated in functional studies on the atria (log Kact, 9.1 ± 0.10). These functional studies also provided estimates of the log Kact and Kinact values of the allosteric agonist LY211,9620 (6.3 and 5.1, respectively), the log isomerization constant of the unoccupied M2 muscarinic receptor (Kq, −3.18 ± 0.11), and the log sensitivity constant of the signaling pathway (log KE, 1.87 ± 0.27). Receptor‐state affinity constants provide a more powerful approach to structure activity relationships and to the measurement of agonist bias.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.