New Mollusk-Specific .alpha.-Conotoxins Block Aplysia Neuronal Acetylcholine Receptors

Abstract

Two mollusc-specific neurotoxic peptides from the venom of the molluscivorous snail Conus pennaceus are described. These new toxins block acetylcholine receptors (AChR) of cultured Aplysia neurons. Bath application of 0.5-1 microM toxin induces 5-10-mV membrane depolarization, which recovers to the control level within 1-3 min in the presence of the toxin. This response is blocked by 1 mM hexamethonium. Concomitantly with the transient depolarization, the toxins block approximately 90% of the depolarizing responses evoked by brief iontophoretic application of acetylcholine. The pharmacology and amino acid sequences of the toxins (alpha PnIA, GCCSLPPCAANNPDYC-NH2; alpha PnIB, GCCSLPPCALSNPDYC-NH2) enable their classification as novel alpha-conotoxins. The sequences differ from those of previously described alpha-conotoxins in a number of features, the most striking of which is the presence of a single negatively charged residue in the C-terminal loop. This loop contains a positively charged residue in piscivorous venom alpha-conotoxins. In contrast to other alpha-conotoxins, which are selective for vertebrate skeletal muscle nicotinic ACh receptors, these Conus pennaceus toxins block neuronal ACh receptors in molluscs. As such they are new probes which can be used to define subtypes of ACh receptors, and they should be useful tools in the study of structure-function relationships in ACh receptors.

Single Amino Acid Substitutions in α-Conotoxin PnIA Shift Selectivity for Subtypes of the Mammalian Neuronal Nicotinic Acetylcholine Receptor

“…In both structures, the side chains of residues 10 and 11 are exposed on the surface of the molecules and hence mutation of these residues would not be expected to produce significant changes in the global fold. Data obtained from 1 H NMR experiments in the current study confirm this is indeed the case. The high degree of surface exposure of these residues and their lack of structural perturbation means that changes in activity among these peptides can be correlated directly to different residue side chains having different binding interactions at different nAChR subtypes.…”

“…These toxins are classified according to their primary structure and biological activity and include the ␣-conotoxin class, which possess a two-loop framework containing two disulfide bonds and are specific inhibitors of nicotinic acetylcholine receptors (nAChRs). 1 Native neuronal nAChRs are composed from a number of distinct subunits (␣2-␣7 and ␣9; ␤2-␤4), which combine to form functional receptors showing a range of pharmacological properties. PnIA and PnIB 2 are 16-residue peptides isolated from the venom of the molluscivorous Conus pennaceus that differ by two amino acids at positions 10 and 11 (see Table I).…”

“…PnIA and PnIB 2 are 16-residue peptides isolated from the venom of the molluscivorous Conus pennaceus that differ by two amino acids at positions 10 and 11 (see Table I). PnIA and PnIB were originally reported to block ACh-evoked responses in Aplysia neurons (1) and, more recently, to exhibit activity in bovine adrenal chromaffin cells, but not at the mammalian neuromuscular junction (2).…”

α-Conotoxin EpI, a Novel Sulfated Peptide from Conus episcopatusThat Selectively Targets Neuronal Nicotinic Acetylcholine Receptors

“…Compared with other 4/7 framework ␣-conotoxins, EpI has homology to both loop one and loop two of PnIA and PnIB and to loop one of MII (see Table I). The ␣-conotoxins EpI, MII (10,43,44), PnIA, PnIB (36), and ImI (9, 43, 45) all contain 4 amino acid residues in loop one, the first of which is serine, suggesting that this construct may be important for neuronal nAChR selectivity. The crystal structure of [Tyr 15 ]EpI and a detailed comparison with PnIA and PnIB are reported elsewhere.…”

Minimal conformation of the ±‐conotoxin ImI for the ±7 neuronalnicotinic acetylcholine receptor recognition: correlated CD, NMR and binding studies

“…1). On the other hand, two c~-conotoxins from Conus pennaceus (c~-PnIA and c~-PnIB) block specifically the neuronal AChR in mollusc [6].…”