Formation of functional α3β4α5 human neuronal nicotinic receptors in Xenopus oocytes:: a reporter mutation approach

1 The alpha5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related beta3 subunit, a5 forms functional recombinant receptors if expressed together with a pair of typical alpha and beta subunits, but its effect on the properties of the resulting alpha beta alpha5 receptor depends on the alpha and beta subunits chosen and on the expression system. We used a reporter mutation approach to test whether alpha5, like beta3, is incorporated as a single copy in human alpha3 beta4 alpha5 receptors expressed in oocytes. 2 As previously reported, the main indication of the presence of alpha5 in alpha3 beta4 alpha5(wt) was an increase in apparent receptor desensitization (compared with alpha3 beta4 receptors). If the alpha3 beta4 alpha5 receptor bore a 9'T mutation in the second transmembrane domain of either alpha3 or beta4, a5 incorporation produced a decrease in ACh sensitivity (by 4 fold for alpha3(LT)beta4 alpha5 vs alpha3(LT)beta4 and by 40 fold for alpha3 beta4(LT)alpha5 vs alpha3 beta4(LT)). The much greater effect observed in alpha3 beta4(LT)alpha5 receptors accords with the hypothesis that alpha5 takes the place of a beta subunit in the receptor. 3 Introducing a 9`T mutation in alpha5 had no effect on the agonist sensitivity of alpha3 beta4 alpha5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. 4 Introducing the 9'T mutation in alpha3 or beta4 in the triplet receptor reduced the EC50 for ACh by a similar extent (7 and 9 fold, respectively), suggesting that alpha3 beta4 alpha5 receptors contain two copies each of alpha and beta and therefore only one copy of alpha5.