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J. Biol. Chem., Vol. 282, Issue 8, 5899-5909, February 23, 2007

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Reversal of Agonist Selectivity by Mutations of Conserved Amino Acids in the Binding Site of Nicotinic Acetylcholine Receptors^*

Nicole A. Horenstein, Thomas J. McCormack¹, Clare Stokes, Ke Ren, and Roger L. Papke²

From the Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610 and the Department of Chemistry, University of Florida, Gainesville, Florida 32611

Homomeric 7 and heteromeric 42 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in 7 receptors for which the 7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in 4 receptors co-expressed with 2 resulted in 42 receptors for which ACh potency was relatively unchanged, although the efficacy of the 7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in 7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both 7 and 4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the 4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.

Received for publication, September 28, 2006 , and in revised form, December 21, 2006.

^* This work was supported by National Institutes of Health Grants R01 GM57481, PO1 AG10485, and T32 AG00196. 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 Present address: Section of Developmental Neurophysiology, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan.

² To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, University of Florida, P. O. Box 100267, Gainesville, FL 32610-0267. Tel.: 352-392-4712; Fax: 352-392-9696; E-mail: rlpapke{at}ufl.edu.

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