α-Conotoxin BuIA, a Novel Peptide from Conus bullatus, Distinguishes among Neuronal Nicotinic Acetylcholine Receptors (original) (raw)

2004, Journal of Biological Chemistry

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. ␣ Subunits, together with ␤2 and/or ␤4 subunits, form ligand-binding sites at ␣/␤ subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the ␣-conotoxin signal sequence and 3-untranslated sequence region, we have cloned a novel gene from the fisheating snail, Conus bullatus; the gene codes for a previously unreported ␣-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, ␣-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for ␣3and chimeric ␣6-containing nAChRs; BuIA blocks ␣6/␣3␤2 nAChRs with a 40,000-fold lower IC 50 than ␣4␤2 nAChRs. The kinetics of toxin unblock are dependent on the ␤ subunit. nAChRs with a ␤4 subunit have very slow off-times, compared with the corresponding ␤2 subunit-containing nAChR. In each instance, rat ␣x␤4 may be distinguished from rat ␣x␤2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse ␣3␤2 to mouse ␣3␤4, and human ␣3␤2 to human ␣3␤4, indicating that the ␤ subunit dependence extends across species. Thus, ␣-conotoxin BuIA also represents a novel probe for distinguishing between ␤2and ␤4-containing nAChRs. Acetylcholine acts on nicotinic acetylcholine receptors (nAChRs) 1 to mediate fast excitatory neurotransmission or to modulate neurotransmitter release. nAChRs appear to be involved in pain sensation, attention, memory, learning, and development (1). Medications that affect nicotinic transmission may be useful for the treatment of pain, memory disorders, Parkinson's disease, schizophrenia, and nicotine addiction. To