Functional Reconstitution and Characterization of Recombinant Human α1-Glycine Receptors

Citation for published article

Metacognitive Strategy Training Is Feasible for People With Aphasia

DOI

10.1074/jbc.M010968200

Document Type

Journal Article

Publication Date

6-15-2001

Publication Title

Journal of Biological Chemistry

School

Bayer School of Natural and Environmental Sciences

Abstract

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 α1-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 α-helical content and a significant fraction of polyproline structure. The small fraction of observed α-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 γ-aminobutyric acid, acetylcholine, and serotonin receptors, may also be erroneously modeled, and alternate models should be considered.

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