Conus venoms are rich sources of biologically active peptides that act specifically on ionic channels and metabotropic receptors present at the neuromuscular junction, efficiently paralyzing the prey. Each species of Conus may have 50 to 200 uncharacterized bioactive peptides with pharmacological interest. Conus regius is a vermivorous species that inhabits Northeastern Brazilian tropical waters. In this work, we characterized one peptide with activity on neuronal acetylcholine receptor (nAChR). Crude venom was purified by reverse-phase HPLC and selected fractions were screened and sequenced by mass spectrometry, MALDI-ToF, and ESI-Q-ToF, respectively. A new peptide was identified, bearing two disulfide bridges. The novel 2,701？Da peptide belongs to the cysteine framework I, corresponding to the cysteine pattern CC-C-C. The biological activity of the purified peptide was tested by intracranial injection in mice, and it was observed that high concentrations induced hyperactivity in the animals, whereas lower doses caused breathing difficulty. The activity of this peptide was assayed in patch-clamp experiments, on nAChR-rich cells, in whole-cell configuration. The peptide blocked slow rise-time neuronal receptors, probably α3β4 and/or α3β4α5 subtype. According to the nomenclature, the new peptide was designated as α-RgIB. 1. Introduction Marine mollusks from Conus genus may produce from 50 up to 200 biologically active molecules that can be injected in the prey to capture or be employed as defense and/or escape mechanisms to deter competitors. The peptide toxins, called conopeptides, are composed of 10–40 amino acids (including nonnatural amino acids) and are abundant in the venom. Peptides presenting a rigid structure due to more than one disulfide bridges are common, being called conotoxins. These peptides act specifically on ionic channels and/or neuromuscular receptors [1, 2]. Conotoxins are classified according to three schemes: the similarities between the endoplasmatic reticulum signal sequence of the conotoxin precursors (gene superfamilies), the cysteine patterns of conotoxin mature peptide regions (cysteine frameworks), and the specificities to pharmacological targets (pharmacological families) [3, 4]. Conopeptides of the pharmacological family α, which acts on neuronal acetylcholine receptor, have been found in the A, D, L, M, and S gene superfamilies [5, 6]. Typically, α-conotoxins are peptides with 12 to 16 amino acid residues and two disulfide bridges, presenting the pattern CC-C-C. These peptides are competitive antagonists of the nicotinic
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