Functional Expression of Human α9*Nicotinic Acetylcholine Receptors in X. laevis Oocytes Is Dependent on the α9 Subunit 5′ UTR

Nicotinic acetylcholine receptors (nAChRs) containing the alpha 9 subunit are expressed in a wide variety of non-neuronal tissues ranging from immune cells to breast carcinomas. The alpha 9 subunit is able to assemble into a functional homomeric nAChR and also co-assemble with the alpha 10 subunit into functional heteromeric nAChRs. Despite the increasing awareness of the important roles of this subunit in vertebrates, the study of human alpha 9-containing nAChRs has been severely limited by difficulties in its expression in heterologous systems. In Xenopus laevis oocytes, functional expression of human alpha 9 alpha 10 nAChRs is very low compared to that of rat alpha 9 alpha 10 nAChRs. When oocytes were co-injected with cRNA of alpha 9 and alpha 10 subunits of human versus those of rat, oocytes with the rat alpha 9 human alpha 10 combination had an similar to-fold higher level of acetylcholine-gated currents (IACh) than those with the human alpha 9 rat alpha 10 combination, suggesting difficulties with human alpha 9 expression. When the ratio of injected human alpha 9 cRNA to human alpha 10 cRNA was increased from 1:1 to 5:1, I-ACh increased 36-fold (from 142 +/- 23 nA to 5171 +/- 748 nA). Functional expression of human alpha 9-containing receptors in oocytes was markedly improved by appending the 5'-untranslated region of alfalfa mosaic virus RNA4 to the 5'-leader sequence of the alpha 9 subunit cRNA. This increased the functional expression of homomeric human alpha 9 receptors by 70-fold (from 7 +/- 1 nA to 475 +/- 158 nA) and of human alpha 9 alpha 10 heteromeric receptors by 80-fold (from 113 +/- 62 nA to 9192 +/- 1137 nA). These findings indicate the importance of the composition of the 5' untranslated leader sequence for expression of alpha 9-containing nAChRs.