Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells

Abstract

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.