1. Academic Validation
  2. Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors

Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors

  • J Biol Chem. 2002 Feb 15;277(7):5433-40. doi: 10.1074/jbc.M109232200.
Brian E Molles 1 Parastoo Rezai Eric F Kline Joseph J McArdle Steven M Sine Palmer Taylor
Affiliations

Affiliation

  • 1 Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA. bmolles@ucsd.edu
Abstract

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the alpha-epsilon than to the alpha-delta binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the alpha-epsilon interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse epsilon subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat epsilon) and 115 (Tyr in mouse, Ser in rat epsilon) converts Wtx-1 affinity for the alpha-epsilon interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both alpha and epsilon subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.

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