Assembly of an adult type acetylcholine receptor in a mouse cell line transfected with rat muscle ϵ-subunit DNA

Abstract

The mouse muscle cell line BC3H-1 expresses an acetylcholine receptor (AChR) composed of α-,β-, and δ-subunits [1]. The functional characteristics of this AChR are comparable to the non-synaptic AChR subtype in mouse muscle [2,3]. To investigate the role of the ϵ-subunit, which is believed to replace the γ-subunit in forming the adult AChR subtype [4], BC3H-1 cells were stably transfected with cDNA encoding the rat muscle AChR ϵ-subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced ϵ-subunit specific RNA after heat induction. Single-channel current recording from cells which contained abundant ϵ-subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle ϵ-subunit may assemble with mouse muscle α-, β- and δ-subunits to form a mouse-rat hybrid AChR with properties similar to that of end-plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in ϵ-subunit mRNA. Thus, the notion that replacement of the γ-subunit by the ϵ-subunit during development is the result of the postnatal rise in the level of ϵ-subunit specific mRNA is further supported.