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J. Biol. Chem., Vol. 282, Issue 44, 32471-32479, November 2, 2007

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Functional Analysis of Transmembrane Domain 2 of the M₁ Muscarinic Acetylcholine Receptor^*

From the Division of Physical Biochemistry, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Ala substitution scanning mutagenesis has been used to probe the functional role of amino acids in transmembrane (TM) domain 2 of the M₁ muscarinic acetylcholine receptor, and of the highly conserved Asn⁴³ in TM1. The mutation of Asn⁴³, Asn⁶¹, and Leu⁶⁴ caused an enhanced ACh affinity phenotype. Interpreted using a rhodopsin-based homology model, these results suggest the presence of a network of specific contacts between this group of residues and Pro⁴¹⁵ and Tyr⁴¹⁸ in the highly conserved NPXXY motif in TM7 that exhibit a similar mutagenic phenotype. These contacts may be rearranged or broken when ACh binds. D71A, like N414A, was devoid of signaling activity. We suggest that formation of a direct hydrogen bond between the highly conserved side chains of Asp⁷¹ and Asn⁴¹⁴ may be a critical feature stabilizing the activated state of the M₁ receptor. Mutation of Leu⁶⁷, Ala⁷⁰, and Ile⁷⁴ also reduced the signaling efficacy of the ACh-receptor complex. The side chains of these residues are modeled as an extended surface that may help to orient and insulate the proposed hydrogen bond between Asp⁷¹ and Asn⁴¹⁴. Mutation of Leu⁷², Gly⁷⁵, and Met⁷⁹ in the outer half of TM2 primarily reduced the expression of functional receptor binding sites. These residues may mediate contacts with TM1 and TM7 that are preserved throughout the receptor activation cycle. Thermal inactivation measurements confirmed that a reduction in structural stability followed the mutation of Met⁷⁹ as well as Asp⁷¹.

Received for publication, May 11, 2007 , and in revised form, August 10, 2007.

^* This work was supported in part by the Medical Research Council, United Kingdom. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S3 and Fig. S1.

¹ Supported by a Medical Research Council post-graduate studentship.

² To whom correspondence should be addressed. Tel.: 44-208-816-2057; Fax: 44-208-906-4477; E-mail: ehulme{at}nimr.mrc.ac.uk.

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