The 3 neuronal nicotinic subunit is localized in dopaminergic areas of the central nervous system, in which many other neuronal nicotinic subunits are expressed. So far, 3 has only been shown to form functional receptors when expressed together with the ␣3 and 4 subunits. We have systematically tested in Xenopus laevis oocytes the effects of coexpressing human 3 with every pairwise functional combination of neuronal nicotinic subunits likely to be relevant to the central nervous system. Expression of ␣7 homomers or ␣/ pairs (␣2, ␣3, ␣4, or ␣6 together with 2 or 4) produced robust nicotinic currents for all combinations, save ␣62 and ␣64. Coexpression of wild-type 3 led to a nearly complete loss of function (measured as maximum current response to acetylcholine) for ␣7 and for all functional ␣/ pairs except for ␣34. This effect was also seen in hippocampal neurons in culture, which lost their robust ␣7-like responses when transfected with 3. The level of surface expression of nicotinic binding sites (␣34, ␣42, and ␣7) in tsA201 cells was only marginally affected by 3 expression. Furthermore, the dominant-negative effect of 3 was abolished by a valine-serine mutation in the 9Ј position of the second transmembrane domain of 3, a mutation believed to facilitate channel gating. Our results show that incorporation of 3 into neuronal nicotinic receptors other than ␣34 has a powerful dominant-negative effect, probably due to impairment in gating. This raises the possibility of a novel regulatory role for the 3 subunit on neuronal nicotinic signaling in the central nervous system.
BackgroundThe neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and β4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or β2 subunits. We compared the properties of human α3β4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α∶β subunit ratio.Methodology/Principal FindingsTwo distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4β2 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 α4β2 channels, for α3β4 receptors the putative two-α form is the predominant one in oocytes (at 1∶1 α∶β cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1∶1 α∶β DNA ratio or in oocytes at 9∶1 α∶β RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS).Conclusions/SignificanceLike other neuronal nicotinic receptors, the α3β4 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.
Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking α (α2–α4, α6) and β (β2, β4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (β4_α2, β4_α3, β4_α4, and β2_α4) when coexpressed with the corresponding β monomer subunit. All tandems expressed when injected alone, except for β4_α3, which produced functional channels only together with β4 monomer and was chosen for further characterization. These channels produced from β4_α3 tandem constructs plus β4 monomer were identical with receptors expressed from monomer α3 and β4 constructs in acetylcholine sensitivity and in the number of α and β subunits incorporated in the channel gate. However, separately mutating the β subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of β subunits from the tandem and one from the β monomer construct, whereas the rest incorporated two or three β monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.
Much of our understanding of ligand-gated ion channels comes from heterologous expression studies. However, this technique cannot produce receptors with a predetermined subunit composition for channels formed by several different subunits and cannot insert a single mutation copy if the subunit of interest is present in several copies in the channel. Here, we describe a novel approach that overcomes these problems by expressing pentameric constructs, in which the code of the five subunits is linked (i.e., 4_4_␣3_4_␣3). This is the first time that a concatemer of the complete pentameric receptor has been expressed for channels in the cysteine-loop superfamily. The presence of the linker did not change the agonist or antagonist sensitivity of ␣34 nicotinic receptors. We show evidence that the expressed receptors were made up of ␣3 and 4 subunits in one pentameric fusion protein as designed in the construct. This approach can be applied to any nicotinic superfamily receptor to produce channels with a defined subunit arrangement and to introduce specific mutations at any desired location of the pentameric fusion protein.
We compared the main properties of human recombinant ␣343 neuronal nicotinic receptors with those of ␣34 receptors, expressed in Xenopus oocytes. 3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (␣34) to 46.7 picosiemens (␣343; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of 3-containing receptors differed little from those of ␣34. The main pharmacological difference in ␣343 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine > > lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2-1 M) and dihydro--erythroidine (30 M) was similar for the two combinations, with a Schild K B for trimetaphan of 76 and 66 nM on ␣34 and ␣343, respectively. The change in single channel conductance confirms that 3 replaces a 4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the 3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of 3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.Among neuronal nicotinic subunits, 3 was long considered an "orphan" as it does not form functional recombinant receptors if expressed as a classical heteromeric combination, i.e.together with either an ␣ or a  subunit. Because of the sequence similarity between 3 and ␣5 we tested the hypothesis that, just like ␣5, 3 would form functional receptors only if co-expressed together with both an ␣ and a  subunit. By a reporter mutation strategy, we showed that 3 is indeed incorporated into functional recombinant ␣343 receptors (1). In ␣343 receptors, 3 is present as a single copy, which replaces one of the  subunits (versus two copies each for the ␣3 and 4 subunits (2)); note that 3 coassembles with other  subunits in rat native cerebellar nAChRs 1 (3). The question now arises of whether the 3 subunit can change the properties of neuronal nicotinic receptors. There are several reasons for investigating this problem. First of all it is important to establish whether any such change introduced by the presence of 3 affects the role that these receptors may have in physiological processes in the central nervous system or in the pharmacology of tobacco addiction. Furthermore, if 3-containing receptors have distinctive biophysical or pharmacol...
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