Human alpha 3 beta 4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

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 [ratio] beta subunit ratio. 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[ratio]1 alpha [ratio] 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[ratio]1 alpha [ratio] beta DNA ratio or in oocytes at 9[ratio]1 alpha [ratio] 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). 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.