Functional renaturation of receptor polypeptides eluted from SDS polyacrylamide gels.
In order to gain further support for the concept that a homo-oligomeric protein-complex may be sufficient to form a functional ligand-activated ion channel and to explore additional possibilities for the reconstitution of channel activity, a single polypeptide band of the purified neuronal AChR from insects has been electroeluted from SDS-polyacrylamide gels, the SDS removed and the polypeptides incorporated into liposomes. Liposomes were fused into planar lipid bilayers which were subsequently analysed for channel activity. Fluctuations of cation-channels were detected after addition of agonists (carbamylcholine); channel activity was blocked by antagonists (d-tubocurarine). The channels formed by electroeluted polypeptides gave conductance values, as well as kinetic data, quite similar to channels formed by the native receptor protein. Sedimentation experiments using sucrose density gradient centrifugation revealed that a considerable portion of the electroeluted polypeptides assembled during the reconstitution process to form oligomeric complexes with a sedimentation coefficient of about 10 S; thus resembling the native receptor complex.
Hanke
W
W
Kernforschungsanlage Jülich, IBI, Federal Republic of Germany.
Andree
J
J
Strotmann
J
J
Kahle
C
C
D000818:Animals; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006110:Grasshoppers / Q000032:analysis*; D007473:Ion Channels / Q000032:analysis*; D008051:Lipid Bilayers / Q000032:analysis; D008081:Liposomes; D010455:Peptides / Q000302:isolation & purification*; D011978:Receptors, Nicotinic / Q000302:isolation & purification*; D014101:Torpedo
D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D010455:Peptides; D011978:Receptors, Nicotinic