Mutagenesis of Nucleophilic Residues near the Orthosteric Binding Pocket of M₁and M₂Muscarinic receptors: Effect on the Binding of Nitrogen Mustard Analogs of Acetylcholine and McN-A-343

Abstract

Investigating how a test drug alters the reaction of a sitedirected electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M 1 and M 2 muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M 1 (Asp105) and M 2 (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M 2 D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M 1 D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M 2 D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M 2 D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M 2 receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M 1 and M 2 receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.

Effects of Asparagine Mutagenesis of Conserved Aspartic Acids in Helix 2 (D2.50) and 3 (D3.32) of M1–M4 Muscarinic Receptors on the Irreversible Binding of Nitrogen Mustard Analogs of Acetylcholine and McN-A-343

“…While this might suggest that BR384 does not alkylate D3.32, and hence, the orthosteric site of M 1 – M 4 receptors, we found that irreversible alkylation of M 1 and M 2 receptors was competitively antagonized by NMS and allosterically inhibited by the allosteric modulator, gallamine (6, 9). We observed the same for the M 2 D3.32N mutant (8), suggesting that BR384 can alkylate a residue in the orthosteric binding pocket of M 1 and M 2 receptors other than D3.32. Although the D3.32N mutation lacked an inhibitory effect on the rate constant for alkylation, it did reduce the affinity of the aziridinium ion to about one-twentieth that of wild type M 1 - M 3 receptors and to about one-fourth that of the wild type M 4 receptor.…”

“…Human embryonic kidney (HEK) 293 cells were cultured as described previously (8) and transfected with plasmids encoding mutated muscarinic receptors using GeneJammer following the manufacturer's protocols. After transfection, the cells were incubated for 48 h and harvested for assays.…”

“…For those experiments involving M 1 D71N/D105N, M 2 D69N/D103N, M 3 D114N/D148N, or M 4 D78N/D112N receptors, a centrifugation assay was used to measure [ 3 H]NMS binding (8) because these mutants exhibited low affinity for [ 3 H]NMS (pK D , 7.7 – 8.0). We were concerned that [ 3 H]NMS-receptor complexes might dissociate during the washing phase of the filtration assay that was used for the other receptors (see below).…”

“…The inhibition was due primarily to a reduction in affinity, however, and not to a decreased rate constant for alkylation, suggesting perhaps that BR384 does not alkylate the D3.32 residue of M 1 and M 2 receptors. Nonetheless, the orthosteric muscarinic antagonist, NMS, competitively inhibited alkylation of wild type M 1 , wild type M 2 and the D103N mutant of the M 2 receptor by BR384, whereas the known allosteric modulator, gallamine, allosterically prevented alkylation (6, 8, 9). Thus, BR384 probably alkylates another residue within the orthosteric-binding pocket of the M 2 receptor.…”

Basic and modern concepts on cholinergic receptor: A review

“…M 2 receptors has indirect role by inhibiting cyclic adenosine monophosphate mediated relaxation in the bladder body [18] , and it has also investigated the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [ 3 H]-Nmethylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex and it was revealed that these toxins, MTα and MTβ, interact with the receptors via kinetically distinct mechanisms [19] . Kimber studied the structure, pharmacology and physiological importance of…”