Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha-9 alpha-10 nicotinic acetylcholine receptor

Halai, Reena, Clark, Richard J., Nevin, Simon T., Jensen, Jonas E., Adams, David J. and Craik, David J. (2009) Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha-9 alpha-10 nicotinic acetylcholine receptor. Journal of Biological Chemistry, 284 30: 20275-20284. doi:10.1074/jbc.M109.015339

Attached Files (Some files may be inaccessible until you login with your UQ eSpace credentials)
Name Description MIMEType Size Downloads
UQ180343_OA.pdf Full text (open access) application/pdf 2.50MB 0

Author Halai, Reena
Clark, Richard J.
Nevin, Simon T.
Jensen, Jonas E.
Adams, David J.
Craik, David J.
Title Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha-9 alpha-10 nicotinic acetylcholine receptor
Formatted title
Scanning mutagenesis of α-conotoxin Vc1.1 reveals residues crucial for activity at the α9α10 nicotinic acetylcholine receptor
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
1083-351X
Publication date 2009-07-24
Year available 2009
Sub-type Article (original research)
DOI 10.1074/jbc.M109.015339
Open Access Status File (Publisher version)
Volume 284
Issue 30
Start page 20275
End page 20284
Total pages 10
Editor Herbert Tabor
Place of publication Bethesda, MD, U.S.A.
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Subject C1
970101 Expanding Knowledge in the Mathematical Sciences
970106 Expanding Knowledge in the Biological Sciences
0601 Biochemistry and Cell Biology
060113 Synthetic Biology
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 alpha 9 alpha 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 alpha 9 alpha 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 alpha 9 alpha 10 and the human alpha 9/rat alpha 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 alpha 3 beta 2, alpha 3 beta 4, and alpha 7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha 9 alpha 10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha 9 alpha 10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.
Formatted 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 α9α10 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 Asp5-Arg7 and Asp11-Ile15, because changes at these positions resulted in the loss of activity at the α9α10 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 α9α10 and the human α9/rat α10 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 α3β2, α3β4, and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.
Keyword Hair-cells
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 41 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 45 times in Scopus Article | Citations
Google Scholar Search Google Scholar
Created: Thu, 03 Sep 2009, 17:47:37 EST by Mr Andrew Martlew on behalf of Institute for Molecular Bioscience