Vol. 286, Issue 2, 619-626, August 1998

Two Domains of the Beta Subunit of Neuronal Nicotinic Acetylcholine Receptors Contribute to the Affinity of Substance P¹

Grace A. Stafford² , Robert E. Oswald, Antonio Figl³ , Bruce N. Cohen³ and Gregory A. Weiland

Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York (G.A.S., R.E.O., G.A.W.) and Division of Biology, California Institute of Technology, Pasadena, California (A.F., B.N.C.)

Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of  and  subunits exhibited differential sensitivity to substance P, with those containing -4 subunits having a 25-fold higher affinity than those having -2 subunits. To identify the regions and/or amino acid residues of the  subunit responsible for this difference, chimeric subunits were coexpressed with -3 in Xenopus oocytes and the IC₅₀ values for substance P were determined. Amino acid residues between 105 and 109 (4 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, 4(V108)/2(F106), which is in the middle of the first extracellular domain, and 4(F255)/2(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  subunit-dependent differences in the receptor affinity for substance P.