Abstract

The β₂-adrenoceptor (β₂AR) fused to the long splice variant of G_sα(G_sαL), but not the β₂AR fused to the short splice variant of G_sα(G_sαS) shows the hallmarks of high constitutive activity, i.e., strong activation of adenylyl cyclase (AC) by GTP and strong inhibition of AC by inverse agonist. These coupling differences are the result of differences in GDP affinity of G_sα splice variants. The aim of this study was to identify experimental variables that differentially affect β₂AR coupling to G_sαS and G_sαL. NaCl substantially reduced agonist-independent AC activation by GTP and inverse agonist inhibition and enhanced agonist stimulation of AC in Sf9 insect cell membranes expressing the β₂AR-G_sαL fusion protein. Salts reduced inverse agonist inhibition and increased agonist stimulation of AC in the order of efficiency NaI ∼ KI > NaBr ∼ KBr > NaCl ∼ LiCl ∼ KCl ∼ RbCl ∼ CsCl ∼ choline chloride, indicating that monovalent anions determine salt effects. Salts inhibited guanosine 5′-O-(3-thiotriphosphate)-mediated AC activation by G_sαL without β₂AR in the order of efficiency NaI > NaBr > NaCl. NaCl enhanced the affinity of G_sαL for GDP. Salts had much smaller effects on β₂AR ligand regulation of AC in membranes expressing β₂AR-G_sαS than in membranes expressing β₂AR-G_sαL. These data are explained by a model in which anions increase the GDP affinity of G_sαL more efficiently than the GDP affinity of G_sαS, and, thereby, decrease the efficiency of the agonist-free β₂AR and increase the efficiency of the agonist-occupied β₂AR at promoting GDP dissociation from G_sαL. Thus, monovalent anions differentially regulate β₂AR-coupling to G_sαS and G_sαL.