Two domains of the beta subunit of neuronal nicotinic acetylcholine receptors contribute to the affinity of substance P.

Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of alpha and beta subunits exhibited differential sensitivity to substance P, with those containing beta-4 subunits having a 25-fold higher affinity than those having beta-2 subunits. To identify the regions and/or amino acid residues of the beta subunit responsible for this difference, chimeric beta subunits were coexpressed with alpha-3 in Xenopus oocytes and the IC50 values for substance P were determined. Amino acid residues between 105 and 109 (beta4 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, beta4(V108)/beta2(F106), which is in the middle of the first extracellular domain, and beta4(F255)/beta2(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 beta subunit-dependent differences in the receptor affinity for substance P.