Paul J. Groot-Kormelink

Single-Channel Behavior of Heteromeric α1β Glycine Receptors: An Attempt to Detect a Conformational Change before the Channel Opens

The α1β heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in the adult. Their activation mechanism was investigated by fitting putative mechanisms to single-channel recordings obtained at four glycine concentrations (10-1000 μm) from rat α1β receptors, expressed in human embryonic kidney 293 cells. The adequacy of each mechanism, with its fitted rate constants, was assessed by comparing experimental dwell time distributions, open-shut correlations, and the concentration-open probability (Popen) curve with the predictions of the model. A good description was obtained only if the mechanism had three glycine binding sites, allowed both partially and fully liganded openings, and predicted the presence of open-shut correlations. A strong feature of the data was the appearance of an increase in binding affinity as more glycine molecules bind, before the channel opens. One interpretation of this positive binding cooperativity is that binding sites interact, each site sensing the state of ligation of the others. 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.

Pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 nicotinic acetylcholine receptors: subunit arrangement determines functional expression

Background and purpose:  α4 and β2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (α4)2(β2)3 and (α4)3(β2)2 receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 receptors.

The Activation Mechanism of α1 Homomeric Glycine 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 α1 glycine receptors by fitting different mechanisms simultaneously to sets of sequences of openings at four glycine concentrations (10–1000 μm). 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 Popen 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.

Openings of the Rat Recombinant α1 Homomeric Glycine Receptor as a Function of the Number of Agonist Molecules Bound

The functional properties of rat homomeric α1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat α1 subunit cDNA. Whole-cell dose-response curves gave EC 50 estimates between 30 and 120 μM and a Hill slope of ∼3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 μM) to outside-out patches. Single channel conductances were mostly 60–90 pS, but smaller conductances of ∼40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.

Stoichiometry of human recombinant neuronal nicotinic receptors containing the β3 subunit expressed in Xenopus oocytes

1 The neuronal nicotinic subunit β3 forms functional receptors when co‐expressed with both an α and a β subunit, such as α3 and β4. We examined the subunit stoichiometry of these ‘triplet’α3β4β3 receptors by expression in Xenopus oocytes of the α3, β4 and β3 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 9′) 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 α3β4β3 receptors, the ACh EC50 was decreased by approximately 17‐fold when this mutation was in α3 alone and only by fourfold when β3 alone was mutated. Mutating β4 was equivalent to mutating α3, suggesting that the ‘triplet’ receptor contains one copy of β3 and two copies each of α3 and β4. 4 Mutating β3 and α3 or β3 and β4 reduced the ACh EC50 further, to values two‐ to threefold lower than those seen when only α3 or β4 carried the mutation. 5 In ‘pair’α3β4 receptors (known to contain two α and three β subunits), mutating β4 had a greater effect on the ACh EC50 than mutating α3, in agreement with an α:β ratio of 2:3 and a constant and independent effect of each copy of the mutation. 6 Our results suggest that α3β4β3 neuronal nicotinic receptors contain one copy of β3 and two copies each of α3 and β4 and confirm that in pair α3β4 receptors the α/β subunits are present in a 2:3 ratio.

Formation of functional α3β4α5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach

The α5 subunit participates to the formation of native neuronal nicotinic receptors, particularly in autonomic ganglia. Like the related β3 subunit, α5 forms functional recombinant receptors if expressed together with a pair of typical α and β subunits, but its effect on the properties of the resulting αβα5 receptor depends on the α and β subunits chosen and on the expression system. We used a reporter mutation approach to test whether α5, like β3, is incorporated as a single copy in human α3β4α5 receptors expressed in oocytes. As previously reported, the main indication of the presence of α5 in α3β4α5wt was an increase in apparent receptor desensitization (compared with α3β4 receptors). If the α3β4α5 receptor bore a 9′T mutation in the second transmembrane domain of either α3 or β4, α5 incorporation produced a decrease in ACh sensitivity (by 4 fold for α3LTβ4α5 vs α3LTβ4 and by 40 fold for α3β4LTα5 vs α3β4LT). The much greater effect observed in α3β4LTα5 receptors accords with the hypothesis that α5 takes the place of a β subunit in the receptor. Introducing a 9′T mutation in α5 had no effect on the agonist sensitivity of α3β4α5 receptors, but reduced apparent desensitisation, as judged by the sag in the current response to high agonist concentrations. Introducing the 9′T mutation in α3 or β4 in the triplet receptor reduced the EC50 for ACh by a similar extent (7 and 9 fold, respectively), suggesting that α3β4α5 receptors contain two copies each of α and β and therefore only one copy of α5.

Incorporation of the β3 Subunit Has a Dominant-Negative Effect on the Function of Recombinant Central-Type Neuronal Nicotinic Receptors

The β3 neuronal nicotinic subunit is localized in dopaminergic areas of the central nervous system, in which many other neuronal nicotinic subunits are expressed. So far, β3 has only been shown to form functional receptors when expressed together with the α3 and β4 subunits. We have systematically tested in Xenopus laevis oocytes the effects of coexpressing human β3 with every pairwise functional combination of neuronal nicotinic subunits likely to be relevant to the central nervous system. Expression of α7 homomers or α/β pairs (α2, α3, α4, or α6 together with β2 or β4) produced robust nicotinic currents for all combinations, save α6β2 and α6β4. Coexpression of wild-type β3 led to a nearly complete loss of function (measured as maximum current response to acetylcholine) for α7 and for all functional α/β pairs except for α3β4. This effect was also seen in hippocampal neurons in culture, which lost their robust α7-like responses when transfected with β3. The level of surface expression of nicotinic binding sites (α3β4, α4β2, and α7) in tsA201 cells was only marginally affected by β3 expression. Furthermore, the dominant-negative effect of β3 was abolished by a valine-serine mutation in the 9′ position of the second transmembrane domain of β3, a mutation believed to facilitate channel gating. Our results show that incorporation of β3 into neuronal nicotinic receptors other than α3β4 has a powerful dominant-negative effect, probably due to impairment in gating. This raises the possibility of a novel regulatory role for the β3 subunit on neuronal nicotinic signaling in the central nervous system.

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 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 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 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/Significance Like 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.

Achieving optimal expression for single channel recording: a plasmid ratio approach to the expression of α1 glycine receptors in HEK293 cells

In single-channel recording, optimal yield of kinetic data is achieved if simultaneous activations of more than one channel are few. When recordings are obtained from recombinant channels, it is therefore important to control the level of expression of the channel at the cell surface, while maintaining a high efficiency of transfection. In the present study, we optimised transfection protocols for single-channel recording from recombinant rat alpha 1 glycine receptors expressed in HEK293 cells. High transfection efficiency was achieved with lipofection (up to 70%). Lipofected cells however did not lend themselves to excised patch recording because of seal instability, especially obvious at hyperpolarised holding potentials. High quality excised patch recordings were reliably achieved with the calcium phosphate-DNA coprecipitation method, with transfection efficiencies around 40%. We achieved good control of the level of receptor expression by a plasmid ratio approach which kept the total amount of plasmid transfected constant while varying the ratio between alpha 1-containing plasmid and empty plasmid vector. The maximum amplitude of glycine-evoked currents was reliably dependent on the percentage of alpha 1-containing plasmid. Optimum results for steady-state single channel experiments at low glycine concentrations were obtained with 5% of alpha 1 plasmid DNA in the transfection mix.

Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors.

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.