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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 Thr192 and Asn382 in human M 1 receptors in ligand binding and receptor activation processes, we created and characterized mutant receptors with Thr192 or Asn382 substituted by Ala. 2 HM 1 wild-type (WT) and mutant receptors [HM 1 (Thr192Ala) and HM 1 (Asn382Ala)] were stably expressed in A9 L cells. The K d values for 3 H-(R)-QNB and K i values for other classical muscarinic antagonists were similar at HM 1 (WT) and HM 1 (Thr192Ala) mutant receptors, yet higher at HM 1 (Asn382Ala) mutant receptors. Carbachol exhibited lower potency and ecacy in stimulating PI hydrolysis via HM 1 (Thr192Ala) mutant receptors, and intermediate agonist activity at the HM 1 (Asn382Ala) mutant receptors. 3 The Asn382 residue in TM VI but not the Thr192 residue in TM V of the human M 1 receptor appears to participate directly in antagonist binding. Both Thr192 and Asn382 residues are involved dierentially in agonist binding and/or receptor activation processes, yet the Asn382 residue is less important than Thr192 in agonist activation of M 1 receptors. 4 Molecular modelling studies indicate that substitution of Thr192 or Asn382 results in the loss of hydrogen-bond interactions and changes in the agonist binding mode associated with an increase in hydrophobic interactions between ligand and receptor.

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