Abstract
Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of α and β subunits exhibited differential sensitivity to substance P, with those containing β-4 subunits having a 25-fold higher affinity than those having β-2 subunits. To identify the regions and/or amino acid residues of the β subunit responsible for this difference, chimericβ subunits were coexpressed with α-3 inXenopus oocytes and the IC50 values for substance P were determined. Amino acid residues between 105 and 109 (β4 numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and the intracellular side of M3, were identified as major contributors to the apparent affinity of substance P. The affinity of acetylcholine was only affected by residue changes between 105 and 109. Site-directed mutagenesis revealed two amino acids that are important determinants of the affinity of substance P, β4(V108)/β2(F106), which is in the middle of the first extracellular domain, and β4(F255)/β2(V253), which is within the putative channel lining transmembrane domain M2. However, other residues within these domains must be making subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the β subunit-dependent differences in the receptor affinity for substance P.
Footnotes
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Send reprint requests to: Dr. Gregory A. Weiland, Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
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↵1 This work was supported by Grant BNS-8911782 from the National Science Foundation and by Cooperative State Research, Education, and Extension Service, USDA (Project Number NYC-425–432) to G.A.W. and by Grant RO1 NS 18660 from the National Institutes of Health to R.E.O. G.A.S. was supported by the Cornell Biotechnology Institute and the Pharmaceutical Manufacturers Association Foundation.
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↵2 Current address: Wadsworth Center, Albany, NY 12201-2002.
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↵3 Current address: Division of Biomedical Sciences, University of California at Riverside, Riverside, CA 9521–0121.
- Abbreviations:
- ACh
- acetylcholine
- DHβE
- dihydro-β-erythroidine
- G protein
- heterotrimeric GTP binding protein
- nAChR
- nicotinic acetylcholine receptor
- nBGT
- neuronal bungarotoxin
- NK
- neurokinin
- nH
- Hill coefficient
- SP
- substance P
- TMA
- tetramethylammonium
- Received October 28, 1997.
- Accepted April 1, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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