Scanning Mutagenesis Identifies Amino Acid Side Chains in Transmembrane Domain 5 of the M1 Muscarinic Receptor that Participate in Binding the Acetyl Methyl Group of Acetylcholine
- Division of Physical Biochemistry, Medical Research Council National Institute for Medical Research, London, United Kingdom
Abstract
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.
Footnotes
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Dr. E. C. Hulme, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. E-mail: ehulme{at}nimr.mrc.ac.uk
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↵1 Current address: SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (South), Third Ave., Essex CM19 5AW, UK.
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↵2 Current address: Department of Pharmacology, University College London, London WC1E 6BT, UK.
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This work was supported by the Medical Research Council, United Kingdom. K. Allman and K. M. Page were supported by Medical Research Council studentships.
- Abbreviations:
- TMD
- transmembrane domain
- ACh
- acetylcholine
- AChR
- acetylcholine receptor
- MAChR
- muscarinic acetylcholine receptor
- NMS
- (−)-N-methylscopolamine
- BrACh
- bromoacetylcholine
- MTSET
- N-trimethyl-2-aminoethylmethanethiosulfonate
- MTSES
- 2-sulfonylethylmethanethiosulfonate
- MTSEA
- 2-aminoethylmethanethiosulfonate
- PI
- phosphoinositide
- SH
- sulfhydryl
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- Received October 14, 1999.
- Accepted March 13, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
