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Originally published In Press as doi:10.1074/jbc.M010968200 on January 5, 2001

J. Biol. Chem., Vol. 276, Issue 24, 20981-20988, June 15, 2001
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Functional Reconstitution and Characterization of Recombinant Human alpha 1-Glycine Receptors*

Michael CascioDagger , Scott Shenkel§, Robert L. Grodzicki, Fred J. Sigworth§, and Robert O. Fox||**

From the Dagger  Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, the § Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, the  Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511, and the || Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555-1055

By utilizing a baculoviral expression system described previously (Cascio, M., Schoppa, N. E., Grodzicki, R. L., Sigworth, F. J., and Fox, R. O. (1993) J. Biol. Chem. 268, 22135-22142), functional recombinant homomeric human alpha 1-glycine receptors (GlyR) were overexpressed in insect cell culture, solubilized, purified, and reconstituted into lipid vesicles via gel filtration. Reconstituted GlyR channels were observed to retain native-like activity in single channel recordings of planar bilayers and in flux assays of small unilamellar vesicles, providing evidence that the recombinant homomeric receptor may be functionally reconstituted. This reconstitution is significant in that it indicates that the overexpressed homomeric receptor is an appropriate substrate for subsequent biophysical characterization aimed at the general elucidation of structure-function. Circular dichroism spectroscopy of reconstituted GlyR indicated a low alpha -helical content and a significant fraction of polyproline structure. The small fraction of observed alpha -helix is insufficient to accommodate the four helical transmembrane domains proposed in models for this receptor. By inference, other members of the homologous ligand-gated channel superfamily, which include the ionotropic gamma -aminobutyric acid, acetylcholine, and serotonin receptors, may also be erroneously modeled, and alternate models should be considered.

* This work was supported by the Howard Hughes Medical Institute (to R. O. F.) and National Institutes of Health Grants NS21501 (to F. J. S.), GM51911 (to M. C.), and GM55851 (to R. O. F).The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Dept. of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6.658 Basic Science Bldg., Galveston, TX 77555-0647. Tel.: 409-772-2163; Fax: 409-747-4745; E-mail: fox@bloch.utmb.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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