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J. Biol. Chem., Vol. 282, Issue 12, 9162-9171, March 23, 2007

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Tryptophan-scanning Mutagenesis in the M3 Transmembrane Domain of the Muscle-type Acetylcholine Receptor

A SPRING MODEL REVEALED^*

José David Otero-Cruz, Carlos Alberto Báez-Pagán, Iván Manuel Caraballo-González, and José Antonio Lasalde-Dominicci¹

From the Departments of Chemistry and Biology, University of Puerto Rico, Río Piedras Campus, San Juan P. R. 00931, Puerto Rico

Membrane proteins constitute a large fraction of all proteins, yet very little is known about their structure and conformational transitions. A fundamental question that remains obscure is how protein domains that are in direct contact with the membrane lipids move during the conformational change of the membrane protein. Important structural and functional information of several lipid-exposed transmembrane domains of the acetylcholine receptor (AChR) and other ion channel membrane proteins have been provided by the tryptophan-scanning mutagenesis. Here, we use the tryptophan-scanning mutagenesis to monitor the conformational change of the M3 domain of the muscle-type AChR. The perturbation produced by the systematic tryptophan substitution along the M3 domain were characterized through two-electrode voltage clamp and ¹²⁵I-labeled -bungarotoxin binding. The periodicity profiles of the changes in AChR expression (closed state) and ACh EC₅₀ (open-channel state) disclose two different helical structures; a thinner-elongated helix for the closed state and a thicker-shrunken helix for the open-channel state. The existence of two different helical structures suggest that the conformational transition of the M3 domain between both states resembles a spring motion and reveals that the lipid-AChR interface plays a key role in the propagation of the conformational wave evoked by agonist binding. In addition, the present study also provides evidence about functional and structural differences between the M3 domains of the Torpedo and muscle-type receptors AChR.

Received for publication, August 7, 2006 , and in revised form, January 18, 2007.

^* This work was supported in part by National Institutes of Health Grants 2RO1GM56371-09 and GM08102-27 and University of Puerto Rico Institutional Funds for Research (to J. A. L.-D.), National Institutes of Health-Minority Biomedical Research Support Research Initiative for Scientific Enhancement Grant R25GM61151 (to C. A. B.-P. and J. D. O.-C.), and the National Science Foundation Alliance for Graduate Education and the Professoriate HRD-0302696 (to C. A. B.-P. and J. D. O.-C.). 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 to. Tel.: 787-764-0000 (ext. 1-2765 or 4887); Fax: 787-753-3852; E-mail: joseal{at}coqui.net or jlasalde{at}gmail.com.

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