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

Abstract

The α5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related β3 subunit, α5 forms functional recombinant receptors if expressed together with a pair of typical α and β subunits, but its effect on the properties of the resulting αβα5 receptor depends on the α and β subunits chosen and on the expression system. We used a reporter mutation approach to test whether α5, like β3, is incorporated as a single copy in human α3β4α5 receptors expressed in oocytes. As previously reported, the main indication of the presence of α5 in α3β4α5^wt was an increase in apparent receptor desensitization (compared with α3β4 receptors). If the α3β4α5 receptor bore a 9′T mutation in the second transmembrane domain of either α3 or β4, α5 incorporation produced a decrease in ACh sensitivity (by 4 fold for α3^LTβ4α5 vs α3^LTβ4 and by 40 fold for α3β4^LTα5 vs α3β4^LT). The much greater effect observed in α3β4^LTα5 receptors accords with the hypothesis that α5 takes the place of a β subunit in the receptor. Introducing a 9′T mutation in α5 had no effect on the agonist sensitivity of α3β4α5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. Introducing the 9′T mutation in α3 or β4 in the triplet receptor reduced the EC₅₀ for ACh by a similar extent (7 and 9 fold, respectively), suggesting that α3β4α5 receptors contain two copies each of α and β and therefore only one copy of α5. British Journal of Pharmacology (2001) 134, 789–796; doi:10.1038/sj.bjp.0704313