Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Abstract

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the ␣9␣10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp 5 -Arg 7 and Asp 11 -Ile 15 , because changes at these positions resulted in the loss of activity at the ␣9␣10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K؉N9A, all of which were more potent than Vc1.1 at both the rat ␣9␣10 and the human ␣9/rat ␣10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the ␣3␤2, ␣3␤4, and ␣7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the ␣9␣10 nAChR. These findings provide valuable insight into the interaction of Vc1

Structure and Activity of α-Conotoxin PeIA at Nicotinic Acetylcholine Receptor Subtypes and GABAB Receptor-coupled N-type Calcium Channels

“…It is likely that the proline residue in PeIA will also be critical for activity, but this probably derives from a structural, rather than functional, role of this residue. Previous studies have shown that when this proline is mutated to alanine in Vc1.1, significant perturbations in structure are observed (23). Furthermore, we recently showed that a naturally occurring ␣-conotoxin, LtIa, which does not contain this conserved proline, does not have a well defined structure in solution (32).…”

“…Mutagenesis studies on Vc1.1 (23) and RgIA (31) have shown that the Asp-Pro-Arg motif in loop 1 is crucial for activity at the ␣9␣10 nAChR. However, in PeIA, the equivalent residues are His-Pro-Ala, yet it remains a potent antagonist of the ␣9␣10 nAChR.…”

“…Peptide Synthesis-PeIA was synthesized using methods described previously for Vc1.1 (23). Oxidation of the disulfide bonds was achieved by stirring overnight in 0.1 M ammonium bicarbonate buffer.…”

Conus Venom Peptide Pharmacology

“…Scanning mutagenesis of ␣-conotoxin Vc1.1 revealed that Ser4 and Asn9 are important for ␣9␣10 nAChR potency (Halai et al, 2009), whereas for RgIA, Asp5, Pro6, and Arg7 were shown to be critical for both ␣9␣10 and ␣7 nAChR blockade (Ellison et al, 2008). It is noteworthy that these ␣9␣10-selective ␣-conotoxins are analgesic in different animal models of chronic pain, but there is an uncertainty about the true molecular target responsible for this effect.…”

Embryonic Toxin Expression in the Cone Snail Conus victoriae

“…Therefore, the different subtype specificities of Vc1.2 and Vc1.1 for nAChR binding must arise from the specific amino acid side chain differences in these toxins. Amino acid residues affecting the affinity of Vc1.1 for the ␣9␣10 subtype were determined by scanning mutagenesis (54 55)) indicating that changes in these side chains may abolish binding to these nAChR subtypes (Fig. 5).…”