Desperately seeking subunits: are native 5-HT3 receptors really homomeric complexes?

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Desperately seeking subunits: are native 5-HT3 receptors really homomeric complexes? Stephanie Fletcher and Nicholas M. Barnes The 5-HT3 receptor complex is a ligand-gated ion channel, and is therefore likely to comprise multiple subunits in common with other members of this superfamily. To date, however, only one 5-HT3 receptor subunit, plus an alternatively spliced variant, have been identified. In this article, Stephanie Fletcher and Nicholas Barnes review some of the extensive data in the literature that suggest the presence of other 5-HT3 receptor subunits. This is particularly relevant given the recent demonstration that the 5-HT3 receptor purified from pig brain contains a non-5-HT3A-like protein(s). The 5-HT3 receptor is unique among the family of 5-HT receptors in that it comprises a ligand-gated ion channel. The structural relationship of the 5-HT3 receptor with other members of the superfamily of ligand-gated ion channels (e.g. nicotinic acetylcholine, GABAA and glycine receptors, all of which display multiple structurally distinct subunits) might suggest that structurally distinct subunits also assemble to form 5-HT3 receptor complexes. So far, however, only one 5-HT3 receptor subunit and an alternatively spliced variant have been identified (here termed 5-HT3A and 5-HT3As receptor subunits, respectively1,2). Furthermore, the minimal pharmacological and functional differences between these spliced variants (e.g. Refs 2–4), suggests that their presence does not contribute to the reported diversity of native 5-HT3 receptors (see below). Hence, the presence of additional 5-HT3 receptor subunits remains an attractive speculation.

Pharmacological diversity of the 5-HT3 receptor

S. Fletcher, Research Fellow, and N. M. Barnes, Senior Lecturer, Department of Pharmacology, The Medical School, University of Birmingham, Edgbaston, Birmingham, UK B15 2TT.

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It is now well recognized that early attempts to classify subtypes of the 5-HT3 receptor relied largely on inter-species pharmacological differences5, for which considerable data exist. For example, the guinea-pig and human 5-HT3 receptors display a distinct pharmacology relative to a number of other species (e.g. rat, mouse and rabbit6–9). In addition, 5-HT3 receptors demonstrate species differences in relative central distribution9. Whilst inter-species differences are of interest, clearly the presence of intra-species differences would have a greater impact, not least because selective interaction with individual 5-HT3 receptor subtypes may offer TiPS – June 1998 (Vol. 19) PII: S0165-6147(98)01210-3

T increased therapeutic benefit. However, to date, there is little evidence for pharmacological differences in the 5-HT3 receptor within a species (e.g. Refs 10–12, but see Ref. 13), although this does not discount the presence of additional 5-HT3 receptor subunits that are devoid of binding sites for 5-HT (structural subunits?).

Evidence for additional subunits of the 5-HT3 receptor Electrophysiological studies Electrophysiological studies were amongst the first to suggest the presence of different 5-HT3 receptors within a species. For instance, while the conductance of the 5-HT3 receptor ion channel ranges between 9 and 17 pS for the 5-HT3 receptor expressed in various native tissues, it is sub-pS for the 5-HT3 receptor expressed in most cell lines (e.g. the N18, NCB20 and N1E-115 cell lines), and for either of the 5-HT3 receptor spliced variants in heterologous expression systems (for review, see Ref. 14; Table 1)15–17. This suggests that neither the recombinant 5-HT3A or 5-HT3As receptor nor the 5-HT3 receptor expressed in these cell lines has the full characteristics of the native receptor complex. In addition, the single-channel conductance of the 5-HT3 receptor may vary within a single preparation. For example, Derkach et al.18 demonstrated that in neurones of the guinea-pig myenteric plexus, 5-HT3 receptor-mediated currents occurred with two distinct conductances of 9 and 15 pS, which also displayed different rates of desensitization. Furthermore, the channel-conductance differences between different preparations withstand direct comparison. Thus, Hussy and colleagues15 compared the heterologously expressed murine 5-HT3As receptor with the 5-HT3 receptor expressed by murine superior cervical ganglion (SCG) neurones. Whilst the pharmacological profiles of 5-HT3 receptors in these preparations were essentially identical, they displayed marked differences in channel conductance: 0.4–0.6 pS for the heterologously expressed homomeric receptor, compared with approximately 9 pS for the native 5-HT3 receptor channel in SCG neurones (Table 1; Fig. 1). Furthermore, wholecell noise analysis of the 5-HT3 currents of SCG neurones resulted in a single-channel conductance of 3.4 pS, suggesting that, as well as the 9 pS conductance channels, SCG neurones also expressed 5-HT3 receptors that displayed low conductances15. The authors estimated that a composition of 66% small (0.6 pS) and 33% large (8.9 pS) conductance channels would yield the average unitary conductance of 3.4 pS. Indeed, a similar observation (and explanation) had previously been reported for the 5-HT3 receptor channel in rat SCG neurones by Yang et al.16, who demonstrated a 2.5 pS conductance estimated by whole-cell noise analysis compared with a unitary conductance of 11 pS. Hippocampal neurones from both mice and rats also express 5-HT3 receptors with high conductances (approximately 10 pS; Ref. 17). These studies imply the presence of at least two different 5-HT3 receptor channels. However, in common with the related nicotinic

Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0165 – 6147/98/$19.00

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Table 1. Comparison of the electrophysiological properties of the recombinant 5-HT3As receptor and the 5-HT3 receptor expressed in N1E-115 cells and murine and rat neuronal tissue 5-HT3 receptor preparation

Unitary conductancea

Channel conductanceb Rectification

Refs

Recombinant 5-HT3As receptor (murine origin) N1E-115 cells (murine origin) Murine SCG neurones Rat SCG neurones Rat and mouse hippocampus

n.d.c n.d.c 8.9 pS 11 pS 10 pS

0.4–0.6 pS 0.4–0.6 pS 3.4 pS 2.5 pS n.d.

15d 15 15 16 17

Strong Strong Weak Modest Weak

aDetermined

from direct measurements of single-channel currents. bDetermined by fluctuation analysis. cToo low to be resolved. dIn their original paper, this group used the terminology 5-HT3A to describe the recombinant receptor, although they used the shorter spliced variant of the murine 5-HT3A subunit (i.e. the 5-HT3As subunit). n.d., not determined; SCG, superior cervical ganglion.

receptor, the conductance of the 5-HT3 receptor has been shown to be influenced by post-translational modifications such as phosphorylation19. Hence, differential post-translational modifications may account for the conductance differences associated with 5-HT3 receptors. In addition, significant differences in the voltage dependence of the 5-HT3 receptor channels between SCG neurones and heterologously expressed 5-HT3As receptors have been noted15 (Table 1). This might also suggest structural differences between the 5-HT3 receptor complexes in the two preparations. In the same study, the properties of the 5-HT3 receptor expressed in N1E-115 cells (which are murine in origin) were examined: in common with the heterologously expressed 5-HT3As receptor, but in contrast to the receptor expressed by murine SCG neurones, N1E-115 cells possessed lowconductance channels that displayed strong rectification (Table 1; Fig. 1).

Allosteric modulation of the 5-HT3 receptor

In addition to the recognition site for 5-HT, the 5-HT3 receptor possesses pharmacologically distinct sites that mediate allosteric modulation of the receptor complex. As well as evidence for inter-species differences (for review, see Ref. 20), some evidence is available suggesting the presence of intra-species allosteric differences. Thus, the function of the recombinant murine 5-HT3A receptor expressed in HEK293 cells was enhanced by low (0.3–10 ␮M) concentrations of zinc21, yet zinc has been shown to produce a voltage-dependent inhibition of the 5-HT3 receptor-mediated currents recorded from murine NCB20 cells22, the cell line from which the recombinant receptor used in the studies was isolated. It was suggested, by analogy with the influence of subunit composition upon the sensitivity of GABAA receptors to blockade by zinc ions23, that additional subunits may be present within the 5-HT3 receptor expressed by NCB20 cells. However, since the recombinant 5-HT3A receptor and the 5-HT3 receptor expressed in NCB20 cells display sub-pS conductance; this suggests that the proposed zinc-sensitive subunit(s) does not confer the high conductance associated with some 5-HT3 receptor complexes.

Receptor-purification studies Given the apparent lack of success of sequencehomology screening in identifying additional 5-HT3 receptor subunits, it was clear that a different approach was needed. Thus, the 5-HT3 receptor was purified from a native source, pig cerebral cortex, to apparent homogeneity24. The molecular mass of the receptor complex, determined using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) under nonreducing conditions, was approximately 280 kDa, which further supports a pentameric structure for the receptor25–27. SDS–PAGE separation of the affinity-purified protein in reducing buffer (to break disulphide bonds between associated proteins) resulted in between three and six silver-stained protein bands at apparent molecular masses ranging from 37 to 71 kDa (Table 2). This pattern was similar to that observed for the 5-HT3 receptor purified from NG108-15 cells26 (Table 2). In contrast, Lummis and Martin28 observed only a single distinct band of 55 kDa for the 5-HT3 receptor purified from N1E115 cells, although McKernan et al.27 demonstrated that the 5-HT3 receptor purified from NCB20 cells gave broad bands with apparent molecular masses of 38 and 54 kDa (Table 2). Such broad, indistinct bands may be composed of more than one species arising from heterogeneity in post-translational processing (e.g. glycosylation) or distinct subunits migrating closely. In addition to SDS–PAGE separation of the purified 5-HT3 receptor complex from pig cerebral cortex, which results in multiple bands under reducing conditions, it is significant that not all of these bands gave a positive reaction with an antiserum directed against the putative long intracellular loop of the cloned 5-HT3A receptor subunit24 (Table 2). It is unlikely that these non-immunoreactive proteins represent cleaved 5-HT3A or 5-HT3As receptor subunits which have lost their putative intracellular loop since such potential fragments would be considerably smaller (ⱕ37 kDa) than the non-5-HT3Alike proteins. Hence, these protein bands might represent an additional subunit(s) of the 5-HT3 receptor, which, although not apparently influencing the pharmacology of the ligand-gated ion channel, may affect its conductance. This would also help to explain the

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Fig. 1. Schematic representation of the postulated subunit composition of the 5-HT3 receptor complex. a: Cells expressing 5-HT3A or 5-HT3As receptors; b: N1E-115 neuroblastoma cells; c: native neuronal tissue. Orange subunits represent the 5-HT3A or the 5-HT3As subunit; the black subunit represents the non5-HT3A subunit.

apparent failure to detect pharmacologically different 5-HT3 receptors within the same species. Another possibility is that a non-5-HT3A-like protein co-purifies with the 5-HT3 receptor and may be comparable to either an accessory modulatory protein (such as the 43 kDa protein of the nicotinic receptor, which is involved in receptor clustering29, or the 93 kDa protein gephyrin, which probably anchors the glycine receptor to microtubules in the postsynaptic membrane30), or an endogenous tyrosine kinase (such as the protein that co-purifies

with, and modulates the function of, the NMDA receptor31). Alternatively, the non-5-HT3A-like proteins identified by these purification studies might represent a subunit(s) from another ligand-gated ion channel, since it has recently been reported that the cloned subunit of the 5-HT3 receptor is able to co-assemble with the ␣4 subunit of the nicotinic receptor32. It would be interesting to determine whether native 5-HT3A or 5-HT3As receptors retain this promiscuity (see note added in proof).

Table 2. Protein species associated with purified 5-HT3 receptors from different sources Source of receptor

NCB20 cells N1E-115 cells NG108-15 cells

Pig cerebral cortex

Protein bands Molecular mass (kDa)

Immunoreactivity

38 54 55 36 40 50 76 38 45 50 52 57 60 62 65 71

– + n.d. n.d. n.d. n.d. n.d. + + + – – + – + –

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27 28 26

24

Molecular masses of identified proteins were assessed by sodium dodecyl sulphate-polyacrylamine gel electrophoresis (SDS–PAGE) and their immunoreactivity was assessed using western blotting. + denotes a positive reaction with 5-HT3A antiserum, whilst – denotes no reaction with 5-HT3A antiserum. n.d., not determined.

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V Concluding remarks It remains an attractive hypothesis that the native 5-HT3 receptor complex may contain structurally distinct subunits. Given the established therapeutic actions of 5-HT3 receptor antagonists, the presence of an additional subunit within the 5-HT3 receptor complex would be highly significant: it may add to the diversity of 5-HT3 receptors and hence provide additional targets for drug development, which could also increase the spectrum of therapeutic actions of 5-HT3 receptor ligands.

Note added in proof A recent report failed to find evidence that native 5-HT3 receptors purified from pig brain contain either ␣1, ␣3, ␣4, ␣5, ␣7 or ␤2 subunits of the nicotinic receptor [Fletcher, S., Lindstrom, J. M., McKernan, R. M. and Barnes, N. M. (1998) Neuropharmacology 37, 397–399]. Selected references

1 Maricq, A. V., Peterson, A. S., Brake, A. J., Myers, R. M. and Julius, D. (1991) Science 254, 432–437 2 Hope, A. G. et al. (1993) Eur. J. Pharmacol. 245, 187–192 3 Downie, D. L. et al. (1994) Neuropharmacology 33, 473–482 4 Hargreaves, A. C., Lummis, S. C. R. and Taylor, C. W. (1994) Mol. Pharmacol. 46, 1120–1128 5 Richardson, B. P., Engel, G., Donatsch, P. and Stadler, P. A. (1985) Nature 316, 126–131 6 Newberry, N. R., Cheshire, S. H. and Gilbert, M. J. (1991) Br. J. Pharmacol. 102, 615–620 7 Kilpatrick, G. J. et al. (1991) Neurochem. Int. 19, 389–396 8 Malone, H. M., Peters, J. A. and Lambert, J. J. (1991) Br. J. Pharmacol. 104, 68P 9 Parker, R. M. C., Barnes, J. M., Ge, J., Barber, P. C. and Barnes, N. M.

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(1996) J. Neurol. Sci. 144, 119–127 10 Perren, M. J., Rogers, H., Mason, G. S., Bull, D. R. and Kilpatrick, G. J. (1995) Naunyn-Schmiedeberg’s Arch. Pharmacol. 351, 221–228 11 Ito, H. et al. (1995) Neuropharmacology 34, 631–637 12 Akuzawa, S., Miyake, A., Miyata, K. and Fukutomi, H. (1996) Eur. J. Pharmacol. 296, 227–230 13 Bonhaus, D. W., Wong, E. H. F., Stefanich, E., Kunysz, E. A. and Eglen, R. M. (1993) J. Neurochem. 61, 1927–1932 14 Peters, J. A., Malone, H. M. and Lambert, J. J. (1992) Trends Pharmacol. Sci. 13, 391–397 15 Hussy, N., Lukas, W. and Jones, K. A. (1994) J. Physiol. 481, 311–323 16 Yang, J., Mathie, A. and Hille, B. (1992) J. Physiol. 448, 237–256 17 Jones, K. A. and Surprenant, A. (1994) Neurosci. Lett. 174, 133–136 18 Derkach, V., Surprenant, A. and North, R. A. (1989) Nature 339, 706–709 19 van Hooft, J. A. and Vijverberg, H. P. M. (1995) Recept. Channels 3, 7–12 20 Parker, R. M. C., Bentley, K. R. and Barnes, N. M. (1996) Trends Pharmacol. Sci. 17, 95–99 21 Gill, C. H., Peters, J. A. and Lambert, J. J. (1995) Br. J. Pharmacol. 114, 1211–1221 22 Lovinger, D. M. (1991) J. Neurophysiol. 66, 1329–1337 23 Smart, T., Xie, X. and Krishek, B. J. (1994) Prog. Neurobiol. 42, 393–441 24 Fletcher, S. and Barnes, N. M. (1997) Br. J. Pharmacol. 122, 655–662 25 Boess, F. G., Beroukhim, R. and Martin, I. L. (1995) J. Neurochem. 64, 1401–1405 26 Boess, F. G., Lummis, S. C. R. and Martin, I. L. (1992) J. Neurochem. 59, 1692–1701 27 McKernan, R. M. et al. (1990) J. Biol. Chem. 265, 13572–13577 28 Lummis, S. C. R. and Martin, I. L. (1991) Mol. Pharmacol. 41, 18–23 29 Froehner, S. C., Luetje, C. W., Scotland, P. B. and Patrick, J. (1990) Neuron 5, 403–410 30 Kirsch, J. et al. (1991) J. Biol. Chem. 266, 22242–22245 31 Yu, X. M., Askalan, R., Keil, G. J. and Salter, M. W. (1997) Science 275, 674–678 32 van Hooft, J. A., Spier, A. D., Yakel, J. L., Lummis, S. C. R. and Vijverberg, H. P. M. (1997) Soc. Neurosci. Abstr. 23, 374

Acknowledgements The authors are most grateful to Drs Mary Keen and Austen Spruce for comments on the manuscript. The work in the authors’ laboratory is funded by the Medical Research Council, the Wellcome Trust and the British Pharmacological Society.

Coming soon in TiPS... • Capacitative calcium entry and the regulation of smooth muscle tone • Transfer and expression of recombinant nitric oxide synthase genes in the cardiovascular system • A new role for catecholamines: ontogenesis • The elusive nature of intrinsic efficacy • Induction of cytokine receptors by glucocorticoids: functional and pathological significance • Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration

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