Scanning mutagenesis of α-conotoxin AuIB reveals a critical residue for activity at the α3β4 nicotinic acetylcholine receptor

Abstract

a-Conotoxin AuIB, a disulfide-bonded peptide of 15 amino acids with a 4/6 intercysteine spacing, inhibits the a3b4 nicotinic acetylcholine receptor (nAChR) subtype, which is a predominant subtype in the peripheral nervous system. The ribbon isomer of AuIB has been shown to be more potent than the native AuIB (globular isomer) and to discriminate between stoichiometries of a3b4 nAChRs expressed in Xenopus oocytes. AuIB also inhibits high voltage-activated N-type calcium channels in rat DRG neurons via the activation of G protein-coupled GABAB receptors. Interestingly, a-conotoxin AuIB possesses analgesic activity in vivo and, therefore, may be a potential drug lead for treating chronic and neuropathic pain. In order to develop improved drugs void of side effects, it is necessary to understand the molecular determinants of AuIB binding to its putative targets: GABAB receptor vs. a3b4 nAChR. The aim of the present study was to determine the critical amino acid residues of AuIB responsible for its interaction with a3b4 nAChRs. Alanine scanning mutagenesis of the native AuIB peptide was carried out to construct AuIB alanine-substituted analogues which were tested in Xenopus oocytes expressing rat a3 and b4 subunits. Two-electrode voltage clamp recording was used to assess the effect of AuIB and its analogues (3 mM) on the ACh-evoked current amplitude. Phenylalanine to alanine mutation at position 9 of AuIB abolished inhibition of a3b4 nAChRs, whereas substitution of glycine at position 1 with alanine significantly reduced inhibition (18.0 p 10.5%, n = 3) compared to native AuIB (48.5 p 6.9%, n = 7) (p l 0.05). Mutation of residues other than cysteine and proline, which are known to disrupt the tertiary structure of a-conotoxins, did not significantly reduce the inhibition of ACh-evoked currents compared to native AuIB. Subsequent homology modelling/docking simulation was performed using a homology model of the rat (a3)2(b4)3 nAChR. The results suggest that interaction of AuIB Phe9 with Lys81 and Trp79 on the b4 nAChR subunit may be essential for AuIB binding/interaction on a3b4 nAChR. In conclusion, we have identified phenylalanine at position 9 as the critical residue for specific interaction of AuIB with the a3b4 nAChR. Future studies using site-directed mutagenesis of the b4 subunit are required to further dissect the mechanism of AuIB binding/interaction on a3b4 nAChR.