Functional reconstitution and characterization of recombinant human alpha(1)-glycine receptors

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 cultur e, solubilized, purified, and reconstituted into lipid vesicles via gel fil tration. Reconstituted GlyR channels were observed to retain native-like ac tivity in single channel recordings of planar bilayers and in flux assays o f small unilamellar vesicles, providing evidence that the recombinant homom eric receptor may be functionally reconstituted. This reconstitution is sig nificant 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 spectr oscopy of reconstituted GlyR indicated a low alpha -helical content and a s ignificant fraction of polyproline structure. The small fraction of observe d alpha -helix is insufficient to accommodate the four helical transmembran e domains proposed in models for this receptor. By inference, other members of the homologous ligand-gated channel superfamily, which include the iono tropic gamma -aminobutyric acid, acetylcholine, and serotonin receptors, ma y also be erroneously modeled, and alternate models should be considered.