Indeed, no significant effect was observed when responses at 4uC were compared to those at RT. This is consistent with previous evidence indicating that acetylcholine and 4BP-TQS cause activation of a7 nAChRs through different mechanisms of action. It has been reported previously that exposure of some nAChRs to low temperature for several hours can facilitate more efficient protein folding and assembly. This was first demonstrated for the muscle-type nAChR from the marine ray Torpedo, expressed in cultured mammalian cell lines and can be explained by the fact that proteins in cold water fish have not evolved to fold Eupalinilide-C efficiently at 37uC. Similar effects have been reported for insect nAChRs expressed in cultured cell lines. In fact, even mammalian nAChRs have been reported to fold and assemble more efficiently at lower temperature. However, in contrast to these long-term effects occurring over several hours, it is unlikely that increased efficiency of subunit folding and assembly explains the effects reported here. The effects of temperature on current amplitude examine in this study are both very rapid and are reversible, suggesting that the effects observed are a consequence of a change in the thermodynamic properties of already assembled cell-surface receptors, rather than a change in the efficiency of receptor folding and assembly. In contrast to the experiments Antipyrine examining agonist responses at physiological temperature, the rationale for examining responses of human nAChRs at 4uC is probably less obvious. Although responses measured at 4uC do not have direct physiological relevance for human receptors, the fact that lowering temperature to 4uC has opposing effects on a4b2 and a7 nAChRs provides evidence that these two nAChR subtypes have differing biophysical properties. It is unclear why changes in temperature should have opposing effects on current amplitude in two closely related ion channels or why these effects should be largely or completely abolished by either a single point mutation or by activation by an allosteric agonist, rather than by the conventional orthosteric agonist, acetylcholine. It appears, however, that these effects are not a consequence of changes in the rate of receptor desensitization observed during macroscopic oocyte responses, since changes in temperature were found to have a broadly consistent effect on this parameter. In addition, changes in temperature had a consistent effect on the rate of receptor deactivation after removal of agonist, presumably reflecting changes in the off-rate of agonists from their binding sites. It is possible that receptors can adopt multiple open or desensitized states, and that entry into these various states occurs at different rates and can be affected differently by phenomena such as changes in temperature, mutagenesis or by allosteric modulators.