Functional Reconstitution and Characterization of Recombinant Human α1-Glycine Receptors

Michael Cascio, University of Pittsburgh
Scott Shenkel, Yale School of Medicine
Robert L. Grodzicki, Yale University
Fred J. Sigworth, Yale School of Medicine
Robert O. Fox, UT Medical Branch at Galveston


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.