Blue native PAGE as a useful method for the analysis of the assembly of distinct combinations of nicotinic acetylcholine receptor subunits

• Published in: Journal of receptors and signal transduction Vol. 19; no. 1-4; p. 493
• Main Authors: Nicke, A, Rettinger, J, Mutschler, E, Schmalzing, G
• Format: Journal Article
• Language: English
• Published: England 01.01.1999
• Subjects: Animals; Electrophoresis, Polyacrylamide Gel - methods; Female; In Vitro Techniques; Muscles - metabolism; Oocytes - metabolism; Protein Conformation; Rats; Receptors, Nicotinic - chemistry; Receptors, Nicotinic - genetics; Receptors, Nicotinic - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Xenopus laevis
• ISSN: 1079-9893
• DOI: 10.3109/10799899909036667

Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was studied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was alpha 1 beta 1 gamma delta >> alpha 1 beta 1 gamma > alpha 1 beta 1 delta > alpha 1 gamma delta >> alpha 1 delta >> alpha 1 gamma. alpha 1 and alpha 1 beta 1 were not functional. Protein complexes incorporating a heptahistidyl-tagged alpha 1 subunit were chromatographically purified from digitonin extracts of oocytes and resolved by blue native PAGE. In the absence of any co-expressed nAChR subunit, the majority of alpha 1 formed aggregates. Co-expression of beta 1 had no effect on alpha 1 aggregation, whereas both gamma and delta diminished alpha 1 aggregation in favor of discrete oligomers: alpha 1 formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha 1 gamma was complemented with beta 1 to form a functional alpha 1 beta 1 gamma receptor, a small amount of a pentamer was found besides a prominent alpha 1-His7 beta 1 gamma trimer. Expression of the functional alpha 1 beta 1 delta receptor yielded marked amounts of a pentamer besides dimers and trimers. These results are discussed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 1994), substantiating that blue native PAGE is suited for the investigation of ion channel assembly.