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J. Biol. Chem., Vol. 279, Issue 49, 50923-50929, December 3, 2004

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Muscarinic Toxin 7 Selectivity Is Dictated by Extracellular Receptor Loops^*

Anu Kukkonen, Mikael Peräkylä, Karl E. O. Åkerman, and Johnny Näsman¶

From the A. I. Virtanen Institute for Molecular Sciences, Department of Neurobiology and Department of Chemistry, Kuopio University, FIN-70211 Kuopio, Finland

Muscarinic toxin 7 (MT7) is a mamba venom protein antagonist with extremely high selectivity for the M1 muscarinic acetylcholine receptor. To map the sites for the interaction of MT7 with muscarinic receptors we have used chimeric M1:M3 receptors and site-directed mutagenesis of the M3 and M4 receptor subtypes. Two Glu residues in M1, one in extracellular loop 2 and one in extracellular loop 3, were found to be important for the high affinity binding of MT7. Substitution of the corresponding Lys residues in the M3 receptor with Glu converted the M3 mutant to an MT7 binding receptor, albeit with lower affinity compared with M1. A Phe Tyr substitution in extracellular loop 2 of M3 together with the 2 Glu mutations generated a receptor with an increased MT7 affinity (apparent K_i = 0.26 nM in a functional assay) compared with the M1 receptor (apparent K_i = 1.31 nM). The importance of the identified amino acid residues was confirmed with a mutated M4 receptor constructs. The results indicate that the high selectivity of MT7 for the M1 receptor depends on very few residues, thus providing good prospects for future design and synthesis of muscarinic receptor-selective ligands.

Received for publication, June 9, 2004 , and in revised form, September 15, 2004.

^* This study was funded by The Magnus Ehrnroot Foundation, The Sigrid Jusélius Foundation, and by Academy of Finland Grants 48577 (to M. P.), 205693 (to K. E. O. Å.), and 203746 (to J. N.). 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.

^¶ To whom correspondence should be addressed: A. I. Virtanen Institute for Molecular Sciences, Dept. of Neurobiology, P. O. Box 1627, FIN-70211 Kuopio, Finland. Tel.: 358-17-163657; Fax: 358-17-163030; E-mail: Johnny.Nasman{at}uku.fi.

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