Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M-1 muscarinic receptors

1 Conserved amino acids, such as Thr in transmembrane domains (TM)V and Asn in TM VI of muscarinic receptors, may be important in agonist binding and/ or receptor activation. In order to determine the functional roles of Thr19 2 and Asn382 in human M-1, receptors in ligand binding and receptor activat ion processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala. 2 HM1 wild-type (WT) and mutant receptors [HM1(Thr192Ala) and HM1(Asn382Ala )] were stably expressed in A9 L cells. The K-d values for H-3-(R)-QNB and K-i values for other classical muscarinic antagonists were similar at HM1(W T) and HM1(Thr192Ala) mutant receptors, yet higher at HM1(Asn382Ala) mutant receptors. Carbachol exhibited lower potency and efficacy in stimulating P I hydrolysis via HM1(Thr192Ala) mutant receptors, and intermediate agonist activity at the HM1(Asn382Ala) mutant receptors. 3 The Asn382 residue in TM VI but not the Thr192 residue in TM V of the hum an M-1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved differentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less import ant than Thr192 in agonist activation of M-1 receptors. 4 Molecular modelling studies indicate that substitution of Thr192 or Asn38 2 results in the loss of hydrogen-bond interactions and changes in the agon ist binding mode associated with an increase in hydrophobic interactions be tween ligand and receptor.