Acetylcholine mustard (AChM) is an analogue of acetylcholine (ACh) in which the onium headgroup is replaced by a chemically reactive aziridinium moiety.AChM aziridinium has agonist activity, but, having bound, reacts with and blocks the muscarinic receptor (mAChR) binding site.Purified mAChRs from rat forebrain have been specifically labeled with[%I]AChM.The linkage formed is cleaved by hydroxylamine, is found within cyanogen bromide (CNBr) peptides with molecular masses of approximately 2.4 and 3.9 kDa, and is close to a disulfide-bonded cysteine.Edman degradation reveals a site of label attachment 26 residues C-terminal to a CNBr cleavage site.As in the case of the alkylating antagonist analogue[sH]propylbenzilylcholine mustard, these findings indicate that a conserved aspartic acid residue in transmembrane helix 3 of the d C h R s , Corresponding to Asp-106 (ml sequence), is the site of label attachment.We have provided strong evidence that the alkylating antagonist analogue [3H]propylbenzilylcholine mustard aziridinium ([3H]PrBCM)1 labels a conserved aspartic acid residue in transmembrane helix 3 of the muscarinic acetylcholine receptors corresponding to Asp-105 in the rat and human m l mAChR sequence (1,2).In the present study, we show that the corresponding agonist analogue, [3Hlacetylcholine mustard aziridinium ([3H]AChM) labels the same residue as i3H]PrBCM.Although the AChM aziridinium ion has agonist activity, the alkylated receptor is not constitutively activated.A preliminary account of some of these findings has been presented (3).EXPERIMENTAL PROCEDURES 13H]PrBCM (N-~2,3-~qH~]propyl).N.(2-chloroethyl)-2-~inoethyl benzilate, 40 Ci/mmol) and [3HlAChM (N-rnethyl-N-(2-~hloro-l-[~H~lethyl)-2-aminoethyl acetate, 34.5 Ci/mmol) were obtained by custom tritiation (Amersham Corp.). [3H]AChM was stored in dichloromethane, which was removed by evaporation before the ligand was used.Non-radioactive AChM was synthesized by the method of Jackson and Hirst (41, and its identity was verified by NMR.Its properties were as described (5).
Mutations of the dye gene on the E. coli chromosome result in sensitivity to the dye toluidine blue and, in male cells, cause loss of F pili, producing sterility in conjugation. Compared with its dye+ parent, a strain deleted for dye (delta dye) showed an altered sensitivity to a wide range of dyes and antibiotics which affect different intracellular processes, and hence it appeared likely that the barrier properties of the cell envelope were impaired. Unlike mutants known to be defective in LPS structure, there appeared to be no correlation between the hydrophobicity of the compounds and the sensitivity of the delta dye strain. Moreover there was no difference between dye+ and delta dye strains in their sensitivity to LPS-specific phages, and chemical and GLC analysis of LPS components revealed no difference between the two strains. Examination of outer and inner membrane proteins from isogenic strains having the delta dye deletion and the dye+ gene cloned into the plasmid pACYC184, with or without insertional inactivation of dye by the transposon gamma delta, was performed by SDS-PAGE. This revealed a number of differences in the profile of proteins from both inner and outer membranes, correlated with mutation in the dye gene. The dye gene appears to be identical to the sfrA gene, which has been shown to be required for efficient transcription of the sex factor F. It is therefore proposed that the dye (sfrA) gene product may also control the expression of chromosomal genes coding for envelope proteins.
The exofacial part of transmembrane domain 5 (TMD 5) of the cationic amine-binding subclass of 7-transmembrane receptors is thought to be important in binding the side chain of the agonist. Residues Ile-188 through Ala-196 in TMD 5 of the M1 muscarinic acetylcholine receptor (mAChR) have been studied by Cys- and Ala-scanning mutagenesis. The results are consistent with a helical conformation for this sequence. The positively charged sulfhydryl reagentN-trimethyl-2-aminoethyl methanethiosulfonate reacted selectively with Phe-190 → Cys, Thr-192 → Cys, and Ala-193 → Cys, indicating that the face of TMD 5 accessible from the binding site crevice is consistent with a recent model by Baldwin and colleagues of the transmembrane domain of the 7-transmembrane receptors. In contrast, the acetylcholine derivative bromoacetylcholine reacted selectively with Thr-192 → Cys, which forms the focus of a group of amino acids (Ile-188, Thr-189, Thr-192, Ala-196) whose mutation decreased the binding affinity of the transmitter ACh itself. The center of this patch of residues is offset to one side of the binding pocket, suggesting that a rotation of TMD 5, relative to that implied by the Baldwin model, may be necessary to optimize the anchoring of acetylcholine within the binding site of the M1 mAChR. An induced rotation of TMD 5 could contribute to the formation of the activated state of the receptor.
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