Abstract
The human alpha 1 glycine receptor (GlyR) was expressed in Sf9 insect cells infected with a recombinant Autographa californica nuclear polyhedrosis baculovirus. Previous studies had indicated that transient expression of this subunit in Xenopus oocytes or human kidney cell lines is sufficient to form active agonist-gated chloride channels. Expression of the alpha 1 GlyR protein resulted in functional channels present on the cell surface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 48-kDa protein could be easily visualized on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha 1 GlyR protein was solubilized from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminostrychnine agarose, yielding 0.33 mg/liter of cells. Given the low natural abundance of the native channel, the development of this expression system now provides sufficient purified channel protein for future biochemical and biophysical characterization. Since the glycine receptor shares sequence and structural homology with other members of a ligand-gated channel superfamily, further characterization may establish general rules governing the structure and mechanism of these membrane protein channels.
Highlights
The humana1 glycine receptor (GlyR)was expressed malian nervous tissue (Pfeiffer et al, 1982) and was purified in Sf9 insect cells infected with a recombinant Auto- by affinity chromatography on aminostrychnineagarose
Expression of the a1 GlyR protein 93-kDa peripheral polypetide that binds to subsynaptic turesulted in functional channels present on the cellsurface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings
The a1 GlyR protein was solubilized from a membrane the location of antibody determinants and proteolytic digest fraction of infected Sf9 cells in1%digitonin and 0.1% maps, coupled with hydropathy analysisa, model of the chandeoxycholate and purified by affinity chromatography nel topology hasbeen proposed (Betz, 1992)
Summary
GlyR channels mustbe expressed on the surfacoef the insect cells This number, and even the higher numberof channels suggested by the density estimatefrom the patch recordings, is significantly lower than the final yield of purified a1 GlyR protein (0.33 mg/liter). It should be stressed, that the calculated amount of activatable GlyR channels represents only the fractionof expressed protein which is translocated to the plasma membrane and activated witghlycine. I t has been observed in other studies utilizing the baculovirus systemtooverexpressmembraneproteinsthatsignificant quantities of the expressed proteimnay be targeted incorrectly and maybe present in alternative cellular membranes (Miller, 1988) Both quantitation of the finalpurified nl GlyR and examination of whole-cell lysates The final reporteydield of purified recep- complex and/or the decrease in charge to mass ratio of the tor assumes that no proteiwn as lost in anyof these steps
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