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J. Biol. Chem., Vol. 283, Issue 17, 11625-11632, April 25, 2008

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Amino Acid Residues That Confer High Selectivity of the 6 Nicotinic Acetylcholine Receptor Subunit to -Conotoxin MII[S4A,E11A,L15A]^*

Layla Azam¹, Doju Yoshikami, and J. Michael McIntosh

From the Departments of Biology and Psychiatry, University of Utah, Salt Lake City, Utah 84112

Nicotinic acetylcholine receptors (nAChRs) containing 3 and β2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related 6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog of-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric 6/3β2β3 nAChRs with an IC₅₀ of 1.2 nM; in contrast, its IC₅₀ on the closely related 3β2 as well as non-6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the 3 subunit inserted into the homologous positions of the 6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the 6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the 3 subunit increased the IC₅₀ of the peptide by >1000-fold. Conversely, when these key residues in3 were replaced with those from6, the IC₅₀ decreased by almost 150-fold. Similar effects were seen with other 6-selective conotoxins, suggesting the general importance of these6 residues in conferring selective binding.

Received for publication, December 18, 2007 , and in revised form, February 15, 2008.

^* This work was supported by Kirschstein-National Research Service Award Postdoctoral Fellowship DA 016835 (to L. A.) and by National Institutes of Health Grant MH53631 (to J. M. M.) and Grant GM48677 (to D. Y.). 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. of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112. Tel.: 801-581-5907; Fax: 801-585-5010; E-mail: layla_azam{at}yahoo.com.

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