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

Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was st udied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was a lpha(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)b eta(1) were not functional. Protein complexes incorporating a heptahistidyl -tagged alpha(1) subunit were chromatographically purified from digitonin e xtracts 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 g amma and delta diminished alpha(1) aggregation in favor of discrete oligome rs: alpha(1) formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha(1)gamma was complemented with bet a(1) to form a functional alpha(1)beta(1)gamma receptor, a small amount of a pentamer was found besides a prominent alpha(1)-His(7)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 discus sed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 19 94), substantiating that blue native PAGE is suited for the investigation o f ion channel assembly.