Roles of threonine 192 and asparagine 382 in agonist and antagonist interactions with M₁ muscarinic receptors

Abstract

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.

Comparing Label-Free Biosensors for Pharmacological Screening With Cell-Based Functional Assays

“…5,7,8). Furthermore, the relative potency for the agonist and antagonist pairs are consistent with published results (ACh>CCh for M1 15,16 and M2 17 ; scopolamine>pirenzepine for M1 16,18 and M2 18 ; YP-20 ~R121919 19 ). This suggests that more extensive rank-order potency testing of agonists and antagonists would likely translate across the different instruments; meaning receptor pharmacology for these targets is likely independent of the label-free detection system.…”

Mapping of stereoselective recognition sites on human serum transferrin by capillary electrophoresis and molecular modelling

“…The following method was used for the detailed docking and energy minimization study of tryptophan-esters [13]. The conformation of the transferrin structure (containing the hydrogen atoms) was first processed by an energy minimization procedure of 4000 steps.…”

“…The intermolecular term (E L-Tf ) contains the electrostatic and van der Waals energy terms. The binding energy (E B ) was calculated as an energy difference [13]:…”

“…Docking of small molecules on macromolecules is a well-known procedure in drug-design [7]. Several approaches have been developed for such docking studies [8][9][10][11][12][13] or receptor characterisation [14]. The docking energies reflect the strength of the complexation with secondary interactions and provide a basis for a comparison with the experimental results.…”

Quinazolone-Alkyl-Carboxylic Acid Derivatives Inhibit Transmembrane Ca2+Ion Flux to (+)-(S)-α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid

“…The mirrorimage counterpart was placed in the cavity formed by the relaxed protein structure, such as to mimic a change of the ligand conformation. The ligand binding energy, E B , can be calculated as E B ϭ E(RL) Ϫ E(R opt ) Ϫ E(L opt ), where the terms stand for the energy of the optimized receptor-ligand complex, the isolated receptor and ligand, respectively (Huang et al 1999). The first and third term can easily be evaluated using the Sybyl package, but finding the absolute minimum energy conformation for the receptor (R opt ) is practically impossible.…”