M1毒蕈碱型乙酰胆碱受体配体通过G蛋白亚型的差异性偶联调节cAMP蓄积OA

OBJECTIVE To elucidate the biased coupling characteristics of different M1 muscarinic acetylcholine receptor(M1mAChR)ligands to multiple G protein subtypes and to determine how these profiles relate to intracellular cyclic adenosine monophosphate(cAMP)accumulation and calcium mobi-lization.METHODS Human embryonic kidney 293T(HEK293T)cells transiently expressing human M1mAChR were treated with a concentration series(10-13 to 10-4 mol·L-1)of diverse pharmacological ligands,including carbachol(CCh),acetylcholine(ACh),pilocarpine,iperoxo,McN-A-343,GSK1034702,or VU0453595.Bioluminescence resonance energy transfer 1(BRET1)and TRUPATH BRET2 biosen-sors were used to measure ligand-induced transducer recruitment and activation-linked G-protein rear-rangement.Intracellular cAMP accumulation and calcium mobilization were measured using pGloSensor-22F and Fluo-4 AM,respectively.NF449,pertussis toxin(PTX),and FR900359 were used to assess Gαs-,PTX-sensitive Gαi/o-,and Gαq/11-associated contributions.For the positive allosteric modulator(PAM)condition,VU0453595 was preincubated for 10 min before ACh 10 nmol·L-1 was added.Repre-sentative ligands pilocarpine,iperoxo and GSK1034702 were docked to M1mAChR using AutoDock Vina.RESULTS ACh,CCh,and iperoxo produced broad recruitment and activation responses across the measured sensors.Pilocarpine showed a relative preference for β-arrestin2 recruitment,whereas GSK1034702 retained stronger Gαq/11-associated efficacy than β-arrestin2 efficacy.VU0453595 showed no reliable standalone agonist response,but a VU0453595 concentration series in the presence of ACh 10 nmol·L-1 produced responses in Gαq/11,Gαs,and Gαi1 recruitment and activation.The recruitment profile was Gαq/11-oriented relative to the Gαs and Gαi1 sensors,whereas activation-linked responses were more ligand dependent.PTX reversed the high-concentration inhibitory phase of the pilocarpine cAMP response,supporting a contribution from PTX-sensitive Gαi/o-family signaling.Molecular docking confirmed that these ligands stably bound to M1mAChR(-6.5 to-10.2 kcal·mol-1),primarily via hydrogen bonds with tyrosine106 and tryptophan378.CONCLUSION M1mAChR ligands produce distinct cAMP responses through the differential recruitment and activation of Gαq,Gαs,and Gαi1 subtypes.The PAM VU0453595 depends on an orthosteric agonist to modulate G protein bias and second messen-ger signaling.