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 alpha9alpha10 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 alpha9alpha10 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 alpha9alpha10 and the human alpha9/rat alpha10 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 alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.
Highlights
Channels [4, 5]
They interact with nicotinic acetylcholine receptors,4 of both the muscle and the neuronal type, which have been implicated in a range of neurological disorders varying from Alzheimer disease to addiction (6 – 8)
The results from this study provide valuable insight into the key residues involved in the interaction of Vc1.1 with the ␣9␣10 nicotinic acetylcholine receptor (nAChR) subtype and have the potential to assist in the development of conotoxin analogs as drug leads for the treatment of neuropathic pain [4, 33]
Summary
Synthesis and Cleavage of Mutants—All of the peptide mutants were assembled on rink amide methylbenzhydrylamine resin (Novabiochem) using manual solid-phase peptide synthesis with an in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation procedure for Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Crude peptides were purified by reversed phase-HPLC on a Phenomenex C18 column using a gradient of 0 – 80% B in 80 min, with the eluant monitored at 215/280 nm. Membrane currents were recorded from Xenopus oocytes using a GeneClamp 500B amplifier (Molecular Devices), and an automated workstation with eight channels in parallel, including drug delivery and on-line analysis (OpusXpressTM 6000A workstation, Molecular Devices, Sunnyvale, CA). Both the voltage-recording and the current-injecting electrodes were pulled from borosilicate glass (Harvard Apparatus Ltd., Edenbridge, UK) and had resistances of 0.3–1.5 M⍀ when filled with 3 M KCl. All recordings were conducted at room temperature using a bath solution of ND96, as described above. Peak current amplitude was measured before and after incubation of the peptide [23]
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