Lucia G. Sivilotti

On the nature of partial agonism in the nicotinic receptor superfamily

Partial agonists are ligands that bind to receptors but produce only a small maximum response even at concentrations where all receptors are occupied. In the case of ligand-activated ion channels, it has been supposed since 1957 that partial agonists evoke a small response because they are inefficient at eliciting the change of conformation between shut and open states of the channel. We have investigated partial agonists for two members of the nicotinic superfamily—the muscle nicotinic acetylcholine receptor and the glycine receptor—and find that the open–shut reaction is similar for both full and partial agonists, but the response to partial agonists is limited by an earlier conformation change (‘flipping’) that takes place while the channel is still shut. This has implications for the interpretation of structural studies, and in the future, for the design of partial agonists for therapeutic use.

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.

SENSITIZATION OF HIGH MECHANOTHRESHOLD SUPERFICIAL DORSAL HORN AND FLEXOR MOTOR NEURONES FOLLOWING CHEMOSENSITIVE PRIMARY AFFERENT ACTIVATION

&NA; Nociceptive primary afferents have the capacity to induce a state of increased excitability or central sensitization in dorsal horn neurones. This contributes to the mechanical hypersensitivity (allodynia) which occurs after peripheral tissue injury where low‐mechanothreshold primary afferent activation begins to elicit pain. The relative susceptibility of dorsal horn cells with an apparent exclusive nociceptive input (nociceptive‐specific (NS) or high‐threshold (HT) cells) and those with a convergent input from low‐ and high‐threshold mechanoreceptors (wide‐dynamic‐range (WDR) or multireceptive neurones) to sensitivity changes has been disputed. We have examined whether high‐mechanothreshold neurones in the superficial dorsal and the ventral horn can modify their sensitivity following cutaneous application of the chemical irritant mustard oil. This produced both a prolonged reduction in the mechanical threshold of the cutaneous flexion withdrawal reflex, recorded from semitendinosus &agr;‐motor neurones, and an increase in the activity evoked in these neurones by low‐intensity touch stimuli to the glabrous skin. Eight NS or HT only cells, defined in terms of their cutaneous mechanoreceptive field properties, were recorded in the superficial dorsal horn before and after cutaneous application of mustard oil. Mustard oil was applied outside of the mechanical receptive field of the cells and produced a transient increase in action potential discharge in 4 cells but increased the mechanoreceptive field size in all cells for 30–60 min. Mechanical thresholds declined in 6 cells to levels associated with low‐threshold (LT) and WDR cells, and this was accompanied by recruitment of a novel brush/ touch response in 5 cells. The responses evoked by graded electrical stimulation of the sural nerve were tested in 5 cells. Only 1 cell failed to show any change after mustard oil. In 3 cells, an increase in the response to A‐fibre afferents occurred, a novel A‐fibre response was recruited in 2 cells and the C‐fibre response increased in 2 cells. Cells in the superficial dorsal horn of the rat spinal cord that are normally NS can begin, therefore, to respond to LT primary afferent mechanoreceptors after an increase in central excitability produced by activation of peripheral chemoreceptors. Sensitization of these, as well as of WDR cells, may contribute to the generation of post‐injury mechanical pain and reflex hypersensitivity.

Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single‐channel behaviour.

1. In order to establish the subunit composition of neuronal nicotinic receptors in rat superior cervical ganglia (SCG), their single‐channel properties were compared with those of recombinant receptors expressed in Xenopus oocytes, using outside‐out excised patch recording. 2. The mean main conductance of SCG channels from adult and 1‐day‐old rats was 34.8 and 36.6 pS, respectively. Less frequent openings to lower conductances occurred both as isolated bursts and as events connected to the main level by direct transitions. There was considerable interpatch variability in the values of the lower conductances. 3. Nicotinic receptors from oocytes expressing alpha3beta4 and alpha4beta4 subunits had chord conductances lower than that of SCG neurones (22 pS for alpha3beta4 and 29 pS for alpha4beta4). 4. Prolonged recording from both native and recombinant channels was precluded by ‘run‐down’, i.e. channel activity could be elicited for only a few minutes after excision. Nevertheless, SCG channel openings were clearly seen to occur as short bursts (slowest component, 38 ms), whereas recombinant channels opened in very prolonged bursts of activity, the major component being the slowest (480 ms). 5. Addition of the alpha5 subunit to the alpha3beta4 pair produced channels with a higher conductance than those observed after injection of the pair alone (24.9 vs. 22 pS), suggesting incorporation of alpha5 into the channel. Addition of the beta2 subunit did not change alpha3beta4 single‐channel properties. In one out of fourteen alpha3alpha5beta4 patches, both ganglion‐like, high conductance, short burst openings and recombinant‐type, low conductance, slow burst openings were observed. 6. Channels produced by expression in Xenopus oocytes of neuronal nicotinic subunits present in rat SCG as a rule differ from native ganglion receptors in single‐channel conductance and gross kinetics. While it is possible that an essential nicotinic subunit remains to be cloned, it is perhaps more likely that oocytes either cannot assemble neuronal nicotinic subunits efficiently into channels with the correct composition and stoichiometry, or that they produce post‐translational channel modifications which differ from those of mammalian neurones.

THE ION CHANNEL PROPERTIES OF A RAT RECOMBINANT NEURONAL NICOTINIC RECEPTOR ARE DEPENDENT ON THE HOST CELL TYPE

1 A stable mammalian cell line (L‐α3β4) has been established which expresses the cloned rat neuronal nicotinic acetylcholine receptor (nAChR) subunits α3 and β4, which are the most abundant in autonomic ganglia. Ion channel properties of nAChRs expressed in L‐α3β4 cells were investigated by single‐channel and whole‐cell recording techniques, and compared with both rat α3β4 nAChRs expressed in Xenopus oocytes, and endogenous nicotinic receptors in rat superior cervical ganglion (SCO) neurones, using identical solutions for all cell types. 2 Acetylcholine (ACh) caused activation of single ion channel currents with a range of amplitudes. Some channels had high conductances (30–40 pS), and relatively brief lifetimes; these resembled the predominant native channel from SCG. Other channels had low conductances (20–26 pS) and long bursts of openings which were quite unlike native channels, but which were similar to channels formed by α3β4 in oocytes. Both types often occurred in the same patch. 3 Cytisine was about 3 times more potent than ACh (low‐concentration potency ratio) in L‐α3β4 cells, which is not dissimilar to the 5‐fold potency ratio found in both SCG and oocytes, whereas 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) was less potent than ACh in some cells (as in the oocyte), but more potent in others (as in SCG). 4 While the channels expressed in L‐α3β4 cells do not mimic exactly those expressed in rat SCG, they differ considerably from the same subunit combination expressed in oocytes. Larger conductance, SCG‐like channels were detected frequently in L‐α3β4, but were rarely, if ever, seen in oocytes injected with α3 and β4 mRNA. Our results indicate that ion channel properties such as single‐channel conductance can be influenced by the choice of heterologous expression system.

Comparison of neuronal nicotinic receptors in rat sympathetic neurones with subunit pairs expressed in Xenopus oocytes.

1. The agonist sensitivity of nicotinic acetylcholine receptors in rat superior cervical ganglion (SCG) neurones was compared with that of cloned receptors expressed in Xenopus oocytes by pairwise injections of alpha 3‐beta 2 or alpha 3‐beta 4 neuronal nicotinic subunit combinations. 2. Agonist responses in rat SCG neurones indicated that cytisine was the most potent agonist and lobeline the least potent (rank order of potency: cytisine > dimethylphenylpiperazinium iodide (DMPP) > nicotine > ACh > carbachol > lobeline). 3. Receptors expressed in oocytes by injection of alpha 3 and beta 2 subunits had a relatively high sensitivity to DMPP and low sensitivity to cytisine (rank order of potency: DMPP > ACh > lobeline > carbachol > nicotine > cytisine), whereas receptors composed of alpha 3 and beta 4 subunits had a high sensitivity to cytisine and low sensitivity to DMPP (rank‐order of potency: cytisine > nicotine approximately ACh > DMPP > carbachol > lobeline). 4. With the exception of responses to DMPP, agonist sensitivity measurements suggest that nicotinic receptors in the rat SCG are composed of alpha 3 and beta 4 subunits. The results are discussed in terms of the receptor subunit mRNAs known to be expressed in the rat SCG and previous evidence of functional heterogeneity of rat SCG nicotinic acetylcholine receptors.

Properties of human glycine receptors containing the hyperekplexia mutation α1(K276E), expressed in Xenopus oocytes

1 Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single‐channel properties of receptors that contain a mutation, α1(K276E), associated with an atypical form of hyperekplexia. 2 Equilibrium concentration‐response curves showed that recombinant human α1(K276E)β receptors had a 29‐fold lower glycine sensitivity than wild‐type α1β receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged. 3 Both wild‐type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively). 4 Channel openings were shorter for the α1(K276E)β mutant than for the wild‐type α1β, with mean overall apparent open times of 0.82 and 6.85 ms, respectively. 5 The main effect of the α1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose‐response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single‐channel bursts, and the lack of an effect of the mutation on the strychnine‐binding site.

Long duration ventral root potentials in the neonatal rat spinal cord in vitro; the effects of ionotropic and metabotropic excitatory amino acid receptor antagonists

Long duration, primary afferent evoked ventral root potentials (VRPs) have been recorded in vitro from hemisected spinal cords prepared from 8-12-day-old rat pups. Single shock stimulation of a dorsal root at stimulus strengths sufficient to recruit C/group IV afferent fibres evoked a long duration (11.9 +/- 1.2 s) ipsilateral VRP in all preparations. This long duration VRP consisted of two components, (i) a slow wave, time to peak 137.0 +/- 5.1 ms, the amplitude of which was reduced to 8.7% of mean control value in the presence of the N-methyl-D-aspartate (NMDA) antagonist D-AP5 (40 microM), (ii) a prolonged wave with a time to peak of 2.0 +/- 0.2 s which was partially resistant to D-AP5 (40 microM). Both the slow and the prolonged waves were unaffected following superfusion with the metabotropic excitatory amino acid (EAA) receptor antagonist L-AP3 (100-200 microM). Low frequency (1-10 Hz) repetitive stimulation (20 s duration) of high threshold dorsal root afferents evoked a temporal summation of synaptic activity which generated a progressively depolarizing VRP. This cumulative VRP was graded with frequency of stimulation (0.89 +/- 0.13 to 1.25 +/- 0.19 mV). The cumulative VRP was followed by a post-stimulus depolarization which outlasted the period of repetitive stimulation by tens of seconds (47.6 +/- 8.4 to 91.2 +/- 19.9 s). In the presence of AP5 the amplitude of the cumulative VRP was depressed to 54.5 +/- 11.5% of control values when low frequency (1.0 Hz) stimulation was used. The proportion of the cumulative VRP resistant to D-AP5 increased as the frequency of stimulation was increased to 10 Hz. The decay time of the post-stimulus depolarization was unaffected by AP5. Neither the amplitude nor the post-stimulus depolarization of the cumulative VRP was affected by 200 microM L-AP3. It is suggested that both an AP5 sensitive and AP5 insensitive potential contribute to the long duration VRP evoked in the neonatal rat spinal cord following single shock high threshold afferent stimulation. Moreover, the AP5 insensitive prolonged depolarization is manifest following sustained low frequency stimuli and higher frequency inputs.

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.

Function and structure in glycine receptors and some of their relatives

In the field of ligand-gated ion channels, recent developments, both in the knowledge of structure and in the measurement of function at the single-channel level, have allowed a sensible start to be made on understanding the relationship between structure and function in these proteins. In this review, the cases of glycine, nicotinic ACh and glutamate receptors are compared and contrasted, and problems such as how binding of agonist causes the channel to open, and why partial agonists are partial, are considered. Some observations, both structural and functional, suggest that more attention needs to be paid to conformational changes that occur before the channel opens. Such changes might account for the interaction found between subunits of the glycine receptor while it is still shut and, perhaps, the agonist-dependent structural changes seen in AMPA receptors. They might also complicate our understanding of the binding-gating problem.