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

Layla Azam,Cheryl Dowell,Maren Watkins,Jerry A Stitzel,Baldomero M Olivera,J Michael Mcintosh

doi:10.1074/jbc.m406281200

Abstract

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. Alpha subunits, together with beta 2 and/or beta 4 subunits, form ligand-binding sites at alpha/beta subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the alpha-conotoxin signal sequence and 3'-untranslated sequence region, we have cloned a novel gene from the fish-eating snail, Conus bullatus; the gene codes for a previously unreported alpha-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, alpha-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for alpha 3- and chimeric alpha 6-containing nAChRs; BuIA blocks alpha 6/alpha 3 beta 2 nAChRs with a 40,000-fold lower IC(50) than alpha 4 beta 2 nAChRs. The kinetics of toxin unblock are dependent on the beta subunit. nAChRs with a beta 4 subunit have very slow off-times, compared with the corresponding beta 2 subunit-containing nAChR. In each instance, rat alpha x beta 4 may be distinguished from rat alpha x beta 2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse alpha 3 beta 2 to mouse alpha 3 beta 4, and human alpha 3 beta2 to human alpha 3 beta 4, indicating that the beta subunit dependence extends across species. Thus, alpha-conotoxin BuIA also represents a novel probe for distinguishing between beta 2- and beta 4-containing nAChRs.

Highlights

Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels. ␣ Subunits, together with ␤2 and/or ␤4 subunits, form ligand-binding sites at ␣/␤ subunit interfaces
We describe the cloning of a gene that encodes a novel ␣-conotoxin that distinguishes among ␣ subunit-containing Nicotinic acetylcholine receptors (nAChRs) and kinetically discriminates between ␤2- and ␤4-containing nAChRs
Recovery from toxin block was markedly slower for ␤4 versus ␤2 subunit-containing nAChRs. This effect is noticeable when nAChRs containing the same ␣ subunit are compared. We further investigated this effect on mouse ␣3␤2 versus mouse ␣3␤4 nAChRs, and human ␣3␤2 and human ␣3␤4 nAChRs

Summary

EXPERIMENTAL PROCEDURES

Identification and Sequencing of a cDNA Clone Encoding ␣-Conotoxin BuIA—cDNA was prepared by a reverse transcription of RNA isolated from the Conus bullatus venom duct as described previously [4]. To apply a pulse of ACh to the oocyte, the perfusion fluid was switched to one containing ACh for 1 s. This was automatically done at intervals of 1–5 min. The ACh pulse was preceded by perfusion with ND96 (for ␣7) or ND96A (all others). For responses in toxin (test responses), the perfusion solution was switched to one containing toxin while maintaining the same interval of ACh pulses. All ACh pulses contained no toxin, for it was assumed that little, if any, bound toxin would have washed away in the brief time (Ͻ2 s) it takes for the responses to peak. The average peak amplitude of three control responses just preceding exposure to toxin were used to normalize the amplitude of each test response to obtain “% response” or “% block.” Each data point of a dose-response curve represents the

Hill slope

RESULTS

DISCUSSION