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 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.
Cascio
M
M
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Shenkel
S
S
Grodzicki
R L
RL
Sigworth
F J
FJ
Fox
R O
RO
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002784:Cholesterol; D006801:Humans; D008024:Ligands; D008051:Lipid Bilayers; D008564:Membrane Potentials; D010713:Phosphatidylcholines; D018009:Receptors, Glycine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D014162:Transfection
D008024:Ligands; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D018009:Receptors, Glycine; D011994:Recombinant Proteins; D002784:Cholesterol