Functional expression of human α9* nicotinic acetylcholine receptors in X. laevis oocytes is dependent on the α9 subunit 5' UTR.

Abstract

Nicotinic acetylcholine receptors (nAChRs) containing the α9 subunit are expressed in a wide variety of non-neuronal tissues ranging from immune cells to breast carcinomas. The α9 subunit is able to assemble into a functional homomeric nAChR and also co-assemble with the α10 subunit into functional heteromeric nAChRs. Despite the increasing awareness of the important roles of this subunit in vertebrates, the study of human α9-containing nAChRs has been severely limited by difficulties in its expression in heterologous systems. In Xenopus laevis oocytes, functional expression of human α9α10 nAChRs is very low compared to that of rat α9α10 nAChRs. When oocytes were co-injected with cRNA of α9 and α10 subunits of human versus those of rat, oocytes with the rat α9 human α10 combination had an ∼-fold higher level of acetylcholine-gated currents (I(ACh)) than those with the human α9 rat α10 combination, suggesting difficulties with human α9 expression. When the ratio of injected human α9 cRNA to human α10 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 α9-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 α9 subunit cRNA. This increased the functional expression of homomeric human α9 receptors by 70-fold (from 7±1 nA to 475±158 nA) and of human α9α10 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 α9-containing nAChRs.