alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.