-Conotoxin OmIA Is a Potent Ligand for the Acetylcholine-binding Protein as Well as 32 and 7 Nicotinic Acetylcholine Receptors * Received for publication, March 29, 2006, and in revised form, June 5, 2006 Published, JBC Papers in Press, June 27, 2006, DOI 10.1074/jbc.M602969200 Todd T. Talley ‡ , Baldomero M. Olivera § , Kyou-Hoon Han ¶ , Sean B. Christensen § , Cheryl Dowell § , Igor Tsigelny ‡ , Kwok-Yiu Ho ‡ , Palmer Taylor ‡ , and J. Michael McIntosh §1 From the ‡ Department of Pharmacology, University of California, La Jolla, California 92093-0636, the § Department of Biology, University of Utah, Salt Lake City, Utah 84112, the ¶ Molecular Anti-Cancer Research Center, Division of Molecular Therapeutics, Korea Research Institute of Bioscience and Biotechnology, Yusong, P. O. Box 115, Daejon, Korea, and the  Department of Psychiatry, University of Utah, Salt Lake City, Utah 84132 The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the -subunit of nicotinic acetylcholine receptors and in partic- ular the homomeric 7 nicotinic receptor. We report the isola- tion and characterization of an -conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the 7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the 32 nAChR indicating that -conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of 32 nAChRs. -Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17- amino-acid, two-disulfide bridge peptide. The ligand is the first -conotoxin with higher affinity for the closely related receptor subtypes, 32 versus 62, and selectively blocks these two subtypes when compared with 22, 42, and 11 nAChRs. Nicotinic acetylcholine receptors (nAChRs) 2 are found in the neuromuscular junction, peripheral nervous and central nerv- ous systems of both invertebrates and vertebrates. These recep- tors play essential roles in mediating synaptic transmission and modulating the release of a variety of neurotransmitters. Differ- ent molecular forms of the nAChR are comprised of homopen- tameric (7 and 9) and heteropentameric (e.g. 32, 42, and 11) arrangements of subunits that have discrete ana- tomical locations and distinct physiological functions. Dys- function or dysregulation of nAChRs is implicated in a variety of neuropsychiatric disease states including schizophrenia, Par- kinson, Alzheimer, depression and nicotine addiction (1). Sev- eral drug discovery programs aim to develop specific drugs that selectively act on subtypes of nAChRs. The AChBP, synthesized in molluskan glial cells, is proposed to function as a modulator of synaptic ACh transmission. ACh, acting on a glial nAChR, induces cellular release of AChBP. AChBP, in turn, binds presynaptically released ACh, acting as a synaptic buffer to dampen synaptic transmission (2). AChBP has sequence similarity (15–28% identity) with subunits of the cysteine loop ligand-gated ion channel family that includes nAChRs, GABA A , GABA C , 5-hydroxytryptophan type 3, and glycine receptors (3). These receptors assemble as heteromeric or homomeric pentamers of subunits, and each subunit has an NH 2 -terminal extracellular ligand-binding domain and four COOH-terminal transmembrane spans that serve as the chan- nel pore-forming domain. AChBP is homologous to the extra- cellular domain of the cysteine-loop family. The crystal struc- ture of AChBP has provided a structural template for examining ligand recognition in nAChRs by spectroscopic analysis of conjugated fluorophores, structural modeling, dynamics of deuterium/hydrogen exchange, computational docking of ligands, and direct monitoring of ligand occupancy by changes in intrinsic Trp fluorescence (4 – 8). Despite several conserved features of the ACh-binding site among different nAChR subtypes and the AChBP, certain ligands are able to discriminate between nAChRs of different subunit composition. The structural basis of nAChR subtype selectivity and the sequence determinants governing specificity are subjects of intense investigation. -Conotoxins are a family of small peptides used by car- nivorous marine snails to envenomate their prey. These peptides are small, disulfide-linked, conformationally con- strained antagonists of nAChRs. They generally target the ligand-binding site of these receptors. Although the fold of their peptide backbone is highly conserved, differences in amino acid side chains lead to a remarkable degree of recep- tor subtype specificity (9, 10). Recently, -conotoxin ImI (11) and an analog of -conotoxin PnIA (PnIA (A10L,D14K)) (12) have been crystallized bound to the AChBP from Aplysia californica. Both of these -conotox- * This work was supported by United States Public Health Service Fellowship NS 043063 (to T. T. T.), the KRIBB Research Initiative Program (to K.-H. H.) and Ministry of Science and Technology of Korea Grant NSM0140233 (to K.-H. H.), National Institutes of Health Grants GM48677 (to B. M. O.), R37- GM18360, UO1-DA019372 (to P. T.), and MH53631 (to J. M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: Dept. Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840. Tel.: 801-585- 3622; E-mail: mcintosh.mike@gmail.com. 2 The abbreviations used are: nAChR, nicotinic acetylcholine receptors; GABA, -aminobutyric acid; GABA A , -aminobutyric acid type A; GABA C , -ami- nobutyric acid type C; HPLC, high performance liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl; CHAPS, 3-[(3-cholamidopropyl)di- methylammonio]-1-propanesulfonic acid. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 34, pp. 24678 –24686, August 25, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 24678 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 34 • AUGUST 25, 2006 by guest on June 10, 2020 http://www.jbc.org/ Downloaded from