科研之友
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摘要
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.
- 出版日期2009-7-24
