Pharmacological Characterization of Human m1 Muscarinic Acetylcholine Receptors with Double Mutations at the Junction of TM VI and the Third Extracellular Domain1
- Center for Drug Design and Development, Department of Medicinal & Biological Chemistry, College of Pharmacy, The University of Toledo, Toledo, Ohio
Abstract
A mutant human m5 receptor containing the mutations of Ser465 to Tyr and Thr466 to Pro showed constitutive activity. By replacing the equivalent Ser388 with Tyr and Thr389 with Pro, we created a mutant human m1 (Hm1) receptor with comparable double mutations. The mutant receptor, Hm1(Ser388Tyr, Thr389Pro), was stably expressed in A9 L cells and displayed enhanced responses to classical muscarinic agonists with significantly increased potencies. Choline, a normal component of growth media, showed an efficacy comparable to acetylcholine and carbachol at Hm1(Ser388Tyr, Thr389Pro) receptors. Methylcarbachol, a selective nicotinic agonist, exhibited partial agonist activity at human m1 wild-type receptors and full agonist activity at Hm1(Ser388Tyr, Thr389Pro) receptors. l-Hyoscyamine inhibited the activities of choline and methylcarbachol. Muscarinic antagonists displayed small reductions in binding affinities, although muscarinic agonists showed greatly increased binding affinities for Hm1(Ser388Tyr, Thr389Pro) receptors. All agonists, including choline and methylcarbachol, showed multiple affinity states at Hm1(Ser388Tyr, Thr389Pro) receptors in the absence of GppNHp. The high affinity binding sites for acetylcholine, arecoline and choline were shifted in the presence of GppNHp. These results suggest that Hm1(Ser388Tyr, Thr389Pro) is conformationally favorable for agonist binding and receptor activation.
Footnotes
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Send reprint requests to: Dr. W. S. Messer, Jr., Center for Drug Design and Development, Department of Medicinal and Biological Chemistry, College of Pharmacy, The University of Toledo, 2801 W. Bancroft St., Toledo, OH 43606-3390.
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↵1 This work was supported by NS 01493, NS 31173 and NS 35127.
- Abbreviations:
- ACh
- acetylcholine
- APE
- arecaidine propargyl ester hydrobromide, DMEM, Dulbecco’s Modified Eagle Media
- FBS
- fetal bovine serum
- GppNHp
- guanylylimidodiphosphate
- Hm1
- human muscarinic acetylcholine receptor subtype 1
- Hm1(Ser388Tyr
- Thr389Pro), Hm1 mutant receptor with the mutations of Ser388 to Tyr and Thr389 to Pro
- Hm5
- human muscarinic acetylcholine receptor subtype 5
- IP
- inositol phosphate
- KH buffer
- Krebs-Henseleit buffer, mAChR, muscarinic acetylcholine receptor
- (R)-QNB
- (R)-3-quinuclidinyl benzilate
- Ne3
- N-terminal region of the third extracellular domain
- TCA
- trichloroacetic acid, TM, transmembrane domain
- WT
- wild-type
- KSHV-GPCR
- G-protein-coupled receptor of Kaposi’s sarcoma-associated herpesvirus
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- Received February 5, 1998.
- Accepted May 13, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
