Abstract
The alpha-conotoxin Vc1.1 is a small disulfide-bonded peptide currently in development as a treatment for neuropathic pain. This study describes the synthesis, determination of the disulfide connectivity, and the determination of the three-dimensional structure of Vc1.1 using NMR spectroscopy. Vc1.1 was shown to inhibit nicotine-evoked membrane currents in isolated bovine chromaffin cells in a concentration-dependent manner and preferentially targets peripheral nicotinic acetylcholine receptor (nAChR) subtypes over central subtypes. Specifically, Vc1.1 is selective for alpha3-containing nAChR subtypes. The three-dimensional structure of Vc1.1 comprises a small alpha-helix spanning residues Pro6 to Asp11 and is braced by the I-III, II-IV disulfide connectivity seen in other alpha-conotoxins. A comparison of the structure of Vc1.1 with other alpha-conotoxins, taken together with nAChR selectivity data, suggests that the conserved proline at position 6 is important for binding, whereas a number of residues in the C-terminal portion of the peptide contribute toward the selectivity. The structure reported here should open new opportunities for further development of Vc1.1 or analogues as analgesic agents.
Highlights
Tel.: 61-7-3346-2019; Fax: 61-7-3346-2029; E-mail: d.craik@imb.uq.edu.au. 3 The abbreviations used are: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; DQF-COSY, double quantum filtered correlated spectroscopy; RP-HPLC, reversed-phase-high performance liquid chromatography; IC50, half-maximal inhibitory concentration; MS, mass spectrometry; have been implicated in a range of disorders, including Alzheimer disease, schizophrenia, depression, and small cell lung carcinoma, and they play a role in analgesia and addiction [2,3,4,5]
The cysteine spacing within the sequence of Vc1.1 suggests that it is a member of the 4/7 subclass of ␣-conotoxins, which includes the extensively studied conotoxins MII, EpI and PnIB, the actual disulfide connectivity of Vc1.1 has yet to be reported
We report the three-dimensional structure of Vc1.1 in solution as determined by NMR spectroscopy and explicitly determine the disulfide connectivity via chemical reduction and MS analysis
Summary
Peptide Synthesis and Oxidative Folding—Vc1.1, vc1a, and [P6O]-Vc1.1 were assembled on 4-methylbenzhydrylamine amide resin (Auspep) by manual solid-phase peptide synthesis using the in situ neutralization/O-benzotriazole-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate protocol for t-butoxycarbonyl chemistry [12]. Membrane currents were recorded from Xenopus oocytes using an automated work station with eight channels in parallel, including drug delivery and on-line analysis (OpusXpressTM 6000A workstation, Axon Instruments Inc.). Both the voltagerecording and current-injecting electrodes were pulled from borosilicate glass (GC150T-15, Harvard Apparatus Ltd.) and had resistances of 0.3–1.5 M⍀ when filled with 3 M KCl. All recordings were conducted at room temperature (20 –23 °C) using a bath solution of ND96 as described above. Sponding to pA2, which in the case of n ϭ 1 corresponds to Ki [22]
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