TEMPERATURE-SENSITIVE EXPRESSION OF ALL-TORPEDO AND TORPEDO-RAT HYBRID ACHR IN MAMMALIAN MUSCLE-CELLS

When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into α2βγδ pentamers only at temperatures lower than 37°C (Claudio, T., W.N. Green, D.S. Hartman, D. Hayden, H.L. Paulson, F.J. Sigworth, S.M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37°C and 26°C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo α alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37°C. These hybrid AChRs can contain either two Torpedo α subunits or one each of rat and Torpedo α, proving that the two α subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with α subunits expressed in fibroblasts indicate that α polypeptides exhibit different conformations at 26° and 37°C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37°C, there is a reduction in the fraction of α subunits that (a) bind the AChR antagonist α-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled α subunit.