An alternative, and novel, explanation is that agonist binding stabilizes a higher affinity form of the receptor that is produced by a conformational change ("flip") that is separate from, and precedes, channel opening. Both the "interaction" scheme and the flip scheme describe our data well, but the latter has fewer free parameters and above all it offers a mechanism for the affinity increase. Distinguishing between the two mechanisms will be important for our understanding of the structural dynamics of activation in the nicotinic superfamily and is important for our understanding of mutations in these receptors.

The glycine receptor mediates fast synaptic inhibition in the spinal cord and brainstem. Its activation mechanism is not known, despite the physiological importance of this receptor and the fact that it can serve as a prototype for other homopentameric channels. We analyzed single-channel recordings from rat recombinant ␣1 glycine receptors by fitting different mechanisms simultaneously to sets of sequences of openings at four glycine concentrations (10 -1000 M). The adequacy of the mechanism and the rate constants thus fitted was judged by examining how well these described the observed dwell-time distributions, open-shut correlation, and single-channel P open dose-response curve. We found that gating efficacy increased as more glycine molecules bind to the channel, but maximum efficacy was reached when only three (of five) potential binding sites are occupied. Successive binding steps are not identical, implying that binding sites can interact while the channel is shut. These interactions can be interpreted in the light of the topology of the binding sites within a homopentamer.

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 ␣6␤2 and ␣6␤4. 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 ␣3␤4. 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 (␣3␤4, ␣4␤2, 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 ␣3␤4 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.

The muscle-type nicotinic acetylcholine receptor (nAChR) is the best characterised of all ligand-gated ion channels. For this receptor, we know both its subunit composition (a, b, d and e in adult muscle) and subunit stoichiometry (two copies of a and one each of b, d and e). Despite the homology between neuronal and muscle receptor subunits and the likely similarity in the pentameric structures of the assembled muscle and neuronal receptors, the stoichiometry and subunit composition of neuronal nAChRs are still open questions. This is because we have to deal not with one, but with several distinct types of native neuronal nAChRs, and also because the number of combinations theoretically possible (starting from the twelve types of neuronal nicotinic subunits cloned, a2 to a10 and b2 to b4) is very high (McGehee & Role, 1995).Some simplifying assumptions can be made, as only some of the possible combinations can form functional channels when expressed in heterologous systems; for instance, in Xenopus oocytes some a subunits (a7 to a9) can form homomeric channels, whereas most of the other a subunits (namely a2-a4 and a6) can form functional channels only if co-expressed with a b subunit (either b2 or b4). The latter type of receptor (which we shall refer to as a 'pair' receptor) is a pentamer with an a:b stoichiometry of 2:3, as shown by radiolabelling and single channel studies on chick a4b2 receptors expressed in oocytes (Anand et al. 1991;Cooper et al. 1991).Nevertheless, extrapolating the 'either homomer or pair' rule to native receptors may be a serious oversimplification; the fact that one (or two) subunits are sufficient to produce a functional nicotinic receptor does not mean that native receptors are necessarily made in this minimally functional fashion, by just one or two types of subunit. For instance, the main synaptic nAChR in chick parasympathetic ciliary ganglia is made up of three or four different subunits, a3, b4 and a5 (with or 1. The neuronal nicotinic subunit b3 forms functional receptors when co-expressed with both an a and a b subunit, such as a3 and b4. We examined the subunit stoichiometry of these 'triplet' a3b4b3 receptors by expression in Xenopus oocytes of the a3, b4 and b3 subunits, either in wild-type form or after insertion of a reporter mutation.2. The mutation chosen was the substitution of a conserved hydrophobic residue in the second transmembrane domain of the subunits (leucine or valine 9fi) with a hydrophilic threonine. In other ion channels within the nicotinic superfamily, this mutation type consistently increases the potency of agonists. In muscle-type nicotinic receptors, the magnitude of this effect is approximately constant for each mutant subunit incorporated.3. In a3b4b3 receptors, the ACh EC 50 was decreased by approximately 17-fold when this mutation was in a3 alone and only by fourfold when b3 alone was mutated. Mutating b4 was equivalent to mutating a3, suggesting that the 'triplet' receptor contains one copy of b3 and two copies each of a3 and b4.4. Mutating b3 ...

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.