FUNCTIONAL EXPRESSION AND PURIFICATION OF A HOMOMERIC HUMAN-ALPHA-1 GLYCINE RECEPTOR IN BACULOVIRUS-INFECTED INSECT CELLS

The human alpha1 glycine receptor (GlyR) was expressed in Sf9 insect c ells infected with a recombinant Autographa californica nuclear polyhe drosis baculovirus. Previous studies had indicated that transient expr ession of this subunit in Xenopus oocytes or human kidney cell lines i s sufficient to form active agonist-gated chloride channels. Expressio n of the alpha1 GlyR protein resulted in functional channels present o n the cell surface of infected Sf9 cells as evidenced by whole-cell pa tch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 4 8-kDa protein could be easily visualized on Coomassie-stained sodium d odecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha1 GlyR protein was solubiliz ed from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminos trychnine agarose, yielding 0.33 mg/liter of cells. Given the low natu ral abundance of the native channel, the development of this expressio n system now provides sufficient purified channel protein for future b iochemical and biophysical characterization. Since the glycine recepto r shares sequence and structural homology with other members of a liga nd-gated channel superfamily, further characterization may establish g eneral rules governing the structure and mechanism of these membrane p rotein channels.