Human α3β4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

Background: The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the alpha 3 and beta 4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the alpha 5 and/or beta 2 subunits. We compared the properties of human alpha 3 beta 4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and alpha:beta subunit ratio. Methodology/Principal Findings: Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the alpha subunit, as reported for alpha 4 beta 2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike alpha 4 beta 2 channels, for alpha 3 beta 4 receptors the putative two-alpha form is the predominant one in oocytes (at 1:1 alpha:beta cRNA ratio). This two-alpha form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-alpha form is the predominant one in HEK cells transfected with a 1:1 alpha:beta DNA ratio or in oocytes at 9:1 alpha:beta RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-alpha form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-alpha form gave rise to short bursts (14 ms) of high conductance (39 pS). Conclusions/Significance: Like other neuronal nicotinic receptors, the alpha 3 beta 4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.