Kainate receptors: subunits, synaptic localization and function

R Acknowledgements I am grateful to D. Massotte and I. Kitchen for critical review of the manuscript. I thankfully acknowledge Dr F. Pattus and Prof. P. Chambon for their constant support, as well as K. Befort, D. Filliol, C. Gavériaux-Ruff, H. Matthes and F. Simonin for excellent interactions and work.

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44 Wolleman, M., Benyhe, S. and Simon, J. (1993) Life Sci. 52, 599–611 45 Richardson, A., Demoliou-Mason, C. and Barnard, E. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10198–10202 46 König, M. et al. (1996) Nature 383, 535–538 47 Rubinstein, M. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3995–4000 48 Kitchen, I., Slowe, S. J., Matthes, H. W. D. and Kieffer, B. (1997) Brain Res. 778, 73–78 49 Brady, L. S. et al. (1996) Soc. Neurosci. Abstr. 22, 959 50 Ueda, H. et al. (1997) Neurosci. Lett. 237, 136–138

Kainate receptors: subunits, synaptic localization and function Ramesh Chittajallu, Steven P. Braithwaite, Vernon R. J. Clarke and Jeremy M. Henley

R. Chittajallu, Post-doctoral Research Assistant, S. P. Braithwaite, PhD Student, V. R. J. Clarke, Post-doctoral Research Assistant, and J. M. Henley, Reader in Molecular Neuroanatomy, Department of Anatomy, Medical School, University of Bristol, Bristol, UK BS8 1TD.

26

Although it is well established that kainate receptors constitute an entirely separate group of proteins from AMPA receptors, their physiological functions remain unclear. The molecular cloning of subunits that form kainate receptors and the ability to study recombinant receptors is leading to an increased understanding of their functional properties. Furthermore, the development of kainate receptor-selective agonists and antagonists over the past few years is now allowing the physiological roles of these receptors and, in some cases, specific subunits to be investigated. As a consequence, the synaptic activation of postsynaptic kainate receptors and the presence of presynaptic kainate receptors that serve to regulate excitatory and inhibitory synaptic transmission have been described, and will be discussed in this article by Ramesh Chittajallu, Steven Braithwaite, Vernon Clarke and Jeremy Henley. Since the discovery of multiple glutamate receptor subtypes1, major advances in understanding the structures, distributions and roles that these receptors play in the CNS have been made. However, until recently, research into kainate receptors lagged behind that for AMPA and NMDA receptors owing to the lack of suitable pharmacological tools. The purpose of this review is to discuss how recent advances in molecular biology and pharmacology have led to the identification of the possible physiological roles of kainate receptors in the CNS.

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Chemical names CI977: (5R)-(5a,7a,8b-(2)-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro(4,5)dec-8-yl]-4-benzofuranacetamide monohydrochloride) U50488H: (6)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzenacetamide

Recombinant kainate receptors Kainate receptor subunits GluR5 was the first mammalian kainate receptor subunit to be cloned, showing ~40% sequence homology to the AMPA receptor subunits GluR1–GluR4 (Ref. 2). To date, another four kainate receptor subumits (GluR6, GluR7, KA1 and KA2) have been identified. These subunits can be divided into two groups on the basis of their structural homology and affinity for [3H]kainate. The low-affinity subunits, GluR5–GluR7, display 75% homology while the high-affinity subunits, KA1 and KA2, are 68% homologous. The homology between GluR5–GluR7 and KA1/KA2 is much lower at ~45%. As with the AMPA receptor subunits, each of the kainate receptor subunits comprises ~900 amino acids with an Mr of ~100 kDa (Refs 2–9) and are believed to have the same membrane topology10 (Fig. 1). As discussed below, the kainate receptor subunits are subject to both alternative splicing and RNA editing which increase the number of subunit isoforms (Fig. 1).

Alternative splice variants Alternative splicing of GluR5 yields two variants (GluR5-1 and GluR5-2); the former contains an additional 15 amino acids in the extracellular N-terminal region2. Further splice variants of GluR5-2, each possessing one of three alternative C-terminal sequences, have been identified (Fig. 1). The originally identified sequence is designated GluR5-2b, while additional exons located in the C-terminal domain give rise to GluR5-2a and GluR5-2c. This results in the introduction of either a stop codon producing the truncated subunit (GluR5-2a) or an in-frame insertion resulting in the elongated form (GluR5-2c)11. Two C-terminal alternative splice variants of GluR7 (a and b) have also been reported (Fig. 1). The insertion of an additional 40 nucleotide cassette in GluR7b leads to a change in the open reading frame which not only causes an alteration in the amino acid sequence but also an increase in the size of the C-terminal domain. Owing to this splice mechanism, the Cterminal domain of GluR7b possesses no significant sequence homology to other kainate receptor subunits12. As yet, no alternative splicing has been reported for rat GluR6, KA1 or KA2 subunits. However, additional splice variants have been identified for kainate receptor subunits in other species. For example, human GluR5 and mouse

0165-6147/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0165-6147(98)01286-3

R splice site 1

splice site 2

NH2

extracellular

1

2

3

4

membrane bilayer intracellular

COOH

RNA edit site 1

RNA edit site 2

Fig. 1. Topology, alternative splicing and RNA editing of kainate receptor subunits. All kainate receptor subunits share a common topology and possess an extracellular terminus, three transmembrane domains (TM1, TM3 and TM4), with one region which forms a loop within the membrane (M2), leading to an intracellular C-terminal domain10. Variation occurs in subunits encoded by the same gene through RNA editing and alternative splicing at various sites. RNA edit site 1: RNA editing of GluR6 at I/V and Y/C sites in TM1 (Ref. 19). Editing results in a valine at residue 536, whereas unedited RNA encodes for an isoleucine at this position. Further editing in this region leads to a cysteine at residue 540 while unedited RNA encodes for tyrosine at this position. RNA edit site 2: RNA editing of GluR5 (residue 591) and GluR6 (residue 590) at the Q/R site in M2 (Ref. 8). Unedited subunits containing a glutamine residue are designated GluR5(Q) and GluR6(Q), whereas edited subunits, containing an arginine residue at this site are designated GluR5(R) and GluR6(R). Splice site 1: alternative splicing of GluR5 in the N-terminal domain (Ref. 2). GluR5-1 possesses an additional 15 amino acids (890 amino acids); the originally described subunit, GluR5-2b has 875 amino acids. Splice site 2: alternative splicing occurs giving rise to C-terminal splice variants. GluR5-2a possesses a truncated C-terminus (826 amino acids), GluR5-2b is the originally described subunit (875 amino acids), GluR52c has an additional 29 amino acids (904 amino acids11). GluR7 also undergoes alternative splicing giving rise to C-terminal variants12. GluR7a has 888 amino acids, GluR7b contains an additional 55 amino acids (943 amino acids) the frame shift caused results in a very low homology between GluR7a and GluR7b C-termini.

GluR6 both undergo alternative splicing, which leads to changes in the C-terminal sequence. These splice variants are termed GluR5-1d and GluR6-2, respectively13.

Post-transcriptional mRNA editing Like the GluR2 AMPA receptor subunit, GluR5 and GluR6 are subject to RNA editing at the glutamine/ arginine (Q/R) site (Fig. 1)14. The other kainate receptor subunits do not undergo this process and possess a glutamine (Q) residue at this position9. The non-edited form encodes a neutral glutamine (Q) residue in the putative pore-forming second membrane domain (M2), whereas editing confers a positively charged arginine (R) residue at this position. The extent of editing is developmentally regulated in the rat brain. Unlike the GluR2 subunit, however, significant proportions of unedited kainate

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receptor subunits are present in both the embryonic and adult CNS. For example, in the hippocampus, detectable expression of GluR5 and GluR6 mRNA occurs during embryonic development (~E15 and E12, respectively)15. However, although little editing occurs during embryonic stages, 50–60% of GluR5 and 70–95% of GluR6 are edited in adults15–18. For GluR5, the adult pattern of editing in the hippocampus occurs around the fourth postnatal day (P4)17, whereas the majority of GluR6 editing appears to occur earlier, at E14–E19 (Ref. 18). GluR6 can also undergo further editing at two more sites situated in the first hydrophobic domain (Fig. 1)19: isoleucine is changed to valine (I/V), and tyrosine to cysteine (Y/C). This gives the possibility of eight different edited variants of GluR6 subunits and all of them are found to be present to varying extents in the CNS, although the fully edited GluR6(R/V/C) is the most abundantly expressed in the adult CNS (Ref. 19).

Functional characterization of recombinant kainate receptors For all known functional recombinant kainate receptors, kainate elicits a fast onset and rapidly desensitizing response. However, as outlined below, other pharmacological and functional properties differ depending on subunit composition.

Homomeric kainate receptors Of the five kainate receptor subunits, the low-affinity subunits, GluR5 and GluR6, form functional ion channels on homomeric expression displaying a rank order of agonist potency of domoate . kainate .. L-glutamate8,9. It was originally reported that GluR7, KA1 and KA2 do not form functional homomeric channels, despite the latter two having high affinity for [3H]kainate in radioligand binding assays8,9. However, it has recently been shown that GluR7 can form homomeric receptors12 although the potency of agonists is reduced compared with their actions on heteromeric receptors, which suggests that this receptor might have a reduced intrinsic efficacy. Curiously, domoate has no agonist effect even at 100 µM and appears to act as a functional antagonist12. Homomeric GluR5 receptors have been shown to be sensitive to AMPA whereas homomeric receptors comprising either GluR6 or GluR7 are not activated by this compound11,12. The capacity of homomeric GluR5 receptors to be activated by AMPA has been attributed to a single amino acid residue which, when substituted into GluR6, leads to homomeric GluR6 receptors developing an AMPA sensitivity20.

Heteromeric kainate receptors Although the high-affinity subunits KA1 and KA2 fail to form functional homomeric receptors3,4, co-expression with the low-affinity kainate receptor subunits yields functional channels8,9. Given the order of binding affinity for KA1 and KA2, that is kainate . domoate . L-glutamate3,4, one consequence of heteromeric combination with

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Fig. 2. Effects of Q/R editing on the channel properties of homomeric GluR5 and GluR6. a: Introduction of arginine at the Q/R editing site in the second membrane domain (M2) of GluR5 and GluR6 confers a ring of positive charge within the channel pore. This leads to a reduction in single-channel cation conductance (Na1, K1 and Ca21), as indicated by the thickness of the arrows, and thus causing a decrease in unitary conductance of the edited receptor. Conversely, the channel permeability to Cl2 is introduced. Finally, polyamine interaction with the receptor is prevented (note: at physiological pH polyamine molecules are protonated, i.e. positively charged). This leads to changes in the rectification properties of the channel (see b). Thus, unedited homomeric GluR5 and GluR6 display strong inward or double rectification (solid line) whereas edited homomeric GluR5 and GluR6 show an almost linear rectification (dotted line).

low-affinity subunits could be to alter the agonist affinities or their rank order. Indeeed, the pharmacological profiles of homomeric and heteromeric kainate receptors have been shown to differ. For example, although homomeric GluR6 channels are not gated by AMPA, coexpression with KA2 confers AMPA sensitivity21,22. In addition, kainate has a lower potency at GluR6/KA2 receptors when compared to homomeric GluR6 (Ref. 22).

Functional effects of RNA editing of GluR5 and GluR6 Recombinant homomeric receptors comprising unedited kainate receptor subunits [GluR5(Q) or GluR6(Q)] differ in a number of functional characteristics to homomeric receptors that contain edited subunits [GluR5(R) or GluR6(R)]. Thus, homomeric GluR5(R) and GluR6(R) receptors display: (1) a significantly reduced Ca21 permeability23,24; (2) a linear or slightly outwardly rectifying current–voltage relationship instead of the inward or double rectifying properties shown by homomeric GluR5(Q) and GluR6(Q)11,19,23; (3) a single lowconductance state (femtosiemens) rather than the multiple conductance states (picosiemens) detected in the unedited homomeric receptors25; and (4) a highly significant increase in the permeability to Cl2 (Ref. 26). These observations have led to the proposal27 that the ring of positively charged arginine residues within the putative pore-lining hydrophobic domain constitutes an energy barrier to the movement of cations, which would be higher for divalents such as Ca21. Thus, editing would reduce the single-channel conductance and the relative Ca21 permeability and favour the movement of anions such as Cl2 (Fig. 2). Furthermore, the charged arginine residues would 28

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electrostatically prevent polyamines, which are believed to be responsible for the inward or double rectification28,29, from entering the pore on depolarization (Fig. 2). In addition to the functional differences between nonedited homomeric GluR5 and GluR6 receptors with their edited homomeric counterparts, a number of studies have highlighted changes observed in heteromeric assemblies of edited and non-edited subunits. For example, heteromeric receptors comprising GluR5(Q)/GluR5(R) or GluR6(Q)/GluR6(R) produce channels that display a linear or outwardly rectifying current–voltage (I/V) relationship11,19. In addition, GluR6(Q)/GluR6(R) receptors display a significantly reduced Ca21 permeability. Thus, it appears that introduction of edited subunits into receptors that contain unedited subunits is sufficient to elicit similar functional changes to those observed in purely homomeric GluR5(R) and GluR6(R) receptors. However, not all of the functional changes characteristic of edited homomeric receptors occur in such heteromeric assemblies as, for example, GluR6(Q)/GluR6(R) channels remain impermeable to Cl2 (Ref. 26). It has also been observed that co-expression of GluR5(R) or GluR6(R) with KA2 (which is a non-edited subunit) gives rise to channels that have a significantly larger unitary conductance when compared to homomeric GluR5(R) and GluR6(R) channels22,25. Therefore, introduction of a non-edited subunit into homomeric receptors containing edited subunits could act to reverse the functional effects of editing. But again this seems not to be the case for all the functional properties because GluR5(R)/KA2 and GluR6(R)/KA2 channels still display rectification properties that are characteristic of homomeric GluR5(R) and GluR6(R) channels3.

R Although the investigation into the role that Q/R editing plays in altering receptor function has been limited mainly to studies on recombinantly expressed receptors, a similar functional difference occurs in kainate receptors expressed in hippocampal neuronal cultures. Thus, the majority of embryonic hippocampal neurones in culture expressing GluR6(Q) mRNA display kainate receptormediated currents with a marked inward rectification. In contrast, the sole neurone demonstrating linear rectification contained the GluR6(R) transcript30. In summary, it is clear that Q/R editing has a number of consequences for receptor function. However, the mechanism by which this occurs or the exact role that edited subunits play in modifying the function of heteromeric assemblies of native receptors remains to be fully elucidated. Nonetheless, the fact that Q/R editing has been shown to be developmentally regulated15–18 suggests that such modulatory mechanisms might determine the fundamental channel properties of native kainate receptors during development.

Kainate receptor agonists, antagonists and modulators Until recently, the antagonists of choice for non-NMDA receptors have been the quinoxalinediones cyano-7nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX). CNQX and NBQX display a fivefold and 30-fold selective displacement of [3H]AMPA over [3H]kainate binding, respec-

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tively31,32. However, functional studies have shown that CNQX possesses minimal selectivity and NBQX displays only a threefold selectivity between AMPA versus kainate receptor-mediated responses in dorsal root ganglion (DRG) and hippocampal neurones, respectively33–35. Similarly, although CNQX is a very potent inhibitor of kainateinduced non-desensitizing currents (AMPA receptormediated) in cultured hippocampal neurones (IC50 5 0.92 µM), this antagonist also has appreciable antagonist activity (IC50 5 6.1 µM) against kainate-induced desensitizing currents (kainate receptor-mediated) in these neurones36. Agents that modulate the desensitization of AMPA and kainate receptors have been shown previously to display better selectivity. Benzothiadiazides such as cyclothiazide selectively inhibit desensitization of both native and recombinant AMPA receptors with little or no effect on kainate receptors37,38. In contrast, concanavalin A prevents desensitization of native and recombinant kainate receptors with minimal effects at native AMPA receptors37–39. Although concanavalin A is a useful tool in investigating recombinant receptors, or receptors in neuronal cultures, its value in native brain-slice preparations is limited. This is due to a number of technical difficulties arising from its poor tissue penetration and nonspecific binding to carbohydrates and oligosaccharides. A further complication has arisen from the recent observation that concanavalin A can also potentiate, albeit to a lesser extent, kainate- and glutamate-induced currents

Table 1. Agonist pharmacology at native AMPA and kainate receptors Agonist compound

EC50 values at native kainate receptors (mM)a

EC50 values at native AMPA receptors (mM)a

EC50 ratio (AMPA:KA)

Refs

Kainate

6 15 12 22d 23d 14e 0.7 0.6 0.14 0.074 0.011f

160b 120b 64c 240 – – 32b 340c 19 4 325g 240h

27 8 5 11 – – 46 567 136 54 n/a

35 39 44 45 46 47 39 44 48 48 49, 50 51

Domoate ATPAi (S)-5-Iodowillardiine (S)-5-Trifluoromethylwillardiine (2S,4R)-4-Methylglutamatei (SYM2081) aAll

data for native kainate and AMPA receptors were derived from DRG and hippocampal neurones, respectively, unless otherwise stated.

bEC values cited in this study of kainate and domoate are for AMPA receptors in cerebral cortical neurones. 50 cThe EC values of kainate and ATPA are given for AMPA receptor-mediated responses in cerebellar Purkinje neurones. 50 dIn these studies the EC values for kainate refer to those found for kainate receptors in hippocampal neurones and not in DRG. 50 eEC value at kainate receptors in trigeminal neurones. 50 fThe value given here is the IC value for steady-state desensitization induced by a prepulse of SYM2081 to 300 mM kainate in DRG. 50 gEC value at AMPA receptors in neocortical neurones. 50 hEC value at AMPA receptors in cerebellar Purkinje neurones. 50 iRecently it has been shown that ATPA, (S)-5-iodowillardiine and SYM2081 show appreciable selectivity for homomeric GluR5 receptors

over receptors

comprising homomeric assemblies of GluR6 (see Refs 44, 54 and 57). ATPA, (RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid. n/a, to date no EC50 value for SYM2081 has been published for native kainate receptors and therefore no ratio can be calculated.

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Table 2. Antagonist pharmacology at native and recombinant AMPA and kainate receptors Antagonist compound

Antagonism at kainate receptorsa

Antagonism at AMPA receptorsa

Refs

NS-102b

Native: Kb = 6 mM, IC50 = 2.2 mMc Native: IC50 = 1 mM GluR5: IC50 = 2.5 mM GluR6: IC50 >300 mM Native: IC50 = 0.62 mM GluR5: IC50 = 4 mM GluR6 and GluR7: no effect

Native: Kb = 114 mM, IC50 = 4.1 mM Native: IC50 = 0.5 mMe

35 36 52 52 52 44 44 44

LY293558d LY294486d

Native: IC50 >100 mM GluR1–GluR4: IC50 >30 mM

aAll

data for native kainate and AMPA receptors were derived from DRG and hippocampal neurones, respectively, unless otherwise stated. or Kb values for NS-102 at recombinant receptors have not been reported. However, 3 mM NS-102 causes a ~40% and ~10% reduction in kainate(or glutamate-) induced currents in homomeric GluR6 versus heteromeric GluR2/GluR4, respectively. This is in comparison to a 50% and 90% block of GluR6 and GluR2/GluR4-mediated currents, respectively, by 3 mM CNQX (see Ref. 59). cEC50 values for kainate refer to those found for kainate receptors in hippocampal neurones and not in DRG. dNote that, although both LY compounds show selectivity for GluR6 over other kainate receptor subunits, LY294486, unlike LY293558, also displays a high selectivity for kainate over AMPA receptors. eEC50 values in these studies are given for AMPA receptor-mediated responses in cerebellar Purkinje neurones. CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione. bIC 50

in recombinant homomeric GluR1 (AMPA receptor subunit) and NR1-4a (NMDA receptor subunit) receptors40. Recently developed 2,3-benzodiazapines, such as GYKI52466 and GYKI53655 (LY300168) provide selective, noncompetitive antagonism of AMPA receptors41–43. Of the two compounds, GYKI53655 is approximately 10–20-fold more potent than GYKI52466 at native AMPA receptors and at least threefold more potent at homomeric GluR1 and GluR4 receptors34,41,43. Thus, GYKI53655 is currently the most selective AMPA receptor antagonist of choice. Together with the emergence of a new generation of selective kainate receptor agonists and antagonists described below (Tables 1,2; Fig. 3)35,36,39,44–52, these compounds are now permitting distinction between the non-NMDA receptors.

Kainate receptor agonists A compound initially developed as an AMPA receptor agonist, (RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol4-yl)propanoic acid (ATPA)46, displaces [3H]kainate binding from GluR5-containing receptors with a Ki in the low nanomolar range and is more potent than kainate at expressed homomeric GluR5 receptors44. ATPA has Ki values of ~10 mM for displacing [3H]AMPA binding from GluR1–GluR4 and [3H]kainate binding from GluR7 and KA2, with no appreciable activity at GluR6. Halogenated derivatives of willardiine show differing selectivities towards AMPA and kainate receptors. (S)-5Fluorowillardiine is a highly selective AMPA receptor agonist with EC50 values of 1.5 mM in hippocampal neurones (presumed native AMPA receptor-mediated responses) and ~70 mM in the DRG (presumed native kainate receptormediated responses)48,53. (S)-5-Trifluoromethylwillardiine is currently the most potent willardiine at kainate receptors in the DRG, with an EC50 of 74 nM and good selectivity for kainate over AMPA receptors48. However, (S)-5iodowillardiine is the most selective of the willardiine series for kainate receptors with EC50 values of 0.14 mM 30

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in DRG (kainate receptors) and 19 mM in hippocampal neurones (AMPA receptors)48. Interestingly, a recent report has examined the subunit selectivity of (S)-5iodowillardiine for various recombinant kainate receptors54. It elicits desensitizing currents at homomeric GluR5 receptors (EC50 5 83 mM) but has no appreciable activity at homomeric GluR6 or GluR7 receptors and this selectivity has been attributed to a single amino acid in the extracellular region between TM3 and TM4 (Ref. 54). In addition, co-expression of GluR6 with KA2 introduces sensitivity to iodowillardiine (Ref. 54) and this provides further evidence for the differences in the pharmacology of homomeric and heteromeric receptors. A number of diastereomers of 4-methylglutamic acid have been tested for glutamate receptor binding activity. Of the compounds tested, SYM2081 was the most potent inhibitor of [3H]kainate binding and displayed a 3000and 200-fold selectivity at kainate over AMPA and NMDA receptors, respectively55. Like kainate, this compound produces rapidly desensitizing responses in recombinant and native kainate receptors49,50,56. The kainate receptor selectivity of SYM2081 has been further demonstrated by evidence that its EC50 values at homomeric GluR5 and GluR6 are 0.12 mM and 0.23 mM, respectively, whereas at homomeric GluR1 and GluR3, the EC50 values are 132 mM and 453 mM, respectively57. One characteristic feature of kainate receptors is their ability to desensitize at concentrations of agonist that are much lower than those required to elicit currents (for example compare IC50s of ~8 nM and 30 nM for desensitization and EC50s for activation of 1.0 mM and 1.8 mM for SYM2081 and kainate, respectively, at GluR6)49. This property raises the possibility of using such compounds as functional antagonists.

Kainate receptor antagonists The first reported kainate receptor-selective antagonist NS-102 selectively inhibits [3H]kainate binding to

R rat cortical membranes58. NS-102 displays a 20-fold selectivity for kainate versus AMPA receptor-mediated responses35 and preferentially blocks channel currents through recombinant GluR6 kainate receptors compared with GluR2/GluR4 conductances59. In addition, NS-102 inhibits native kainate receptor-mediated responses in a subpopulation of cultured hippocampal neurones45 that have been shown to express high levels of GluR6 (Ref. 30). However, the AMPA receptor selectivity has been questioned because NS-102 inhibits AMPA and kainate receptor-mediated responses in cultured hippocampal neurones with similar IC50 values of 4.1 µM and 2.2 µM, respectively36. These contradicting data about the apparent selectivity of NS-102, together with solubility problems, has meant that this antagonist has been largely superseded. The noncompetitive AMPA receptor antagonist LY293558 also inhibits recombinant kainate receptormediated responses. This effect is highly selective for responses mediated via homomeric GluR5 over homomeric GluR6 receptor-mediated responses52. A second decahydroisoquinoline, LY294486, selectively inhibits [3H]kainate binding to homomeric GluR5 receptors with little effect on recombinant GluR6, GluR7 and KA-2 receptors44. Unlike LY293558 (Ref. 52), LY294486 has an appreciable selectivity towards GluR5 receptors over AMPA receptors44.

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In summary, new-generation compounds such as the selective agonists (S)-5-iodowillardiine and ATPA, and selective antagonists such as GYKI52466, GYKI 53655, LY293558 and LY294486 are providing novel pharmacological tools to allow the differentiation not only between AMPA and kainate receptors but also between individual kainate receptors comprising or containing GluR5 subunits. As described in the following sections, these tools are now beginning to be used to identify the subunit composition and functional roles of native kainate receptors.

Native kainate receptors Kainate receptor subunit distributions Although the exact subunit composition of native kainate receptors is still under investigation (see later discussion), in situ hybridization and immunostaining have indicated the relative distributions of the kainate receptor subunits60–62. Briefly, GluR5 mRNA is limited mainly to the subiculum, CA1 region of the hippocampus and Purkinje cell layer of the cerebellum; GluR6 mRNA is found in high levels primarily in the dentate gyrus and CA3 region of hippocampus, caudate putamen and granule cell layer of cerebellum; GluR7 mRNA displays a widespread, relatively low-level pattern of CH3 COOH H

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COOH CH3 H COOH

CF3

(S )-5-trifluoromethylwillardiine

(2S,4R)-4-methylglutamate (SYM2081)

Fig. 3. Chemical structures of kainate receptor agonists and antagonists. ATPA, (RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid; NS-102, 5-nitro-6,7,8,9-tetrahydrobenzo[g]-2,3-dione-3-oxime.

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distribution; KA1 mRNA is highest in the amygdala, hippocampal formation and entorhinal cortex; KA2 is more widely distributed throughout the CNS with significant levels in cerebral cortex, caudate putamen, hippocampus, entorhinal cortex and granule cell layer of cerebellum. More specifically, within the adult hippocampal formation, differential expression of mRNAs are observed. Transcripts for KA1 and KA2 (KA2 . KA1) are present at the highest levels. KA1 expression is restricted almost exclusively to the CA3 pyramidal and dentate granule neurones compared to an almost equal expression of KA2 in all neurones (CA1–CA3 and dentate gyrus). GluR6 shows a similar expression pattern to KA1 but with a less prominent CA3–CA1 gradient. GluR7 is confined largely to the dentate gyrus and scattered interneurones of the stratum oriens while GluR5 is weakly expressed in areas CA1 and CA3 with little apparent expression in the dentate gyrus60–62. During development, gene expression peaks in the late embryonic period {E12; that is approximately two days before apparent receptor expression as measured by [3H]kainate binding}. The GluR6 CA3–CA1 gradient develops more slowly than KA1 (~P12 and P0, respectively) and appears to remain constant thereafter. This contrasts with a sharply defined peak, restricted to area CA1, in GluR5 expression at stage P0–P5 that begins to decline at P12 to a still detectable but diminished level in adults61. The temporally restricted expression of GluR5 is reflected in spatial changes in [3H]kainate binding observed at this time and appears to be restricted mainly to interneurones within the stratum radiatum and especially the stratum oriens61. In addition to analysis of gene expression, the use of anti-GluR5/6/7, anti-GluR6/7 and anti-KA2 subunit antibodies has localized kainate receptor subunits at postsynaptic membranes and in dendritic spines of pyramidal cells in CA3 and CA1 regions of the hippocampus62–65.

Postsynaptic kainate receptors Peripheral nervous system (PNS) Early evidence for pure kainate receptor-mediated responses came from observations in the PNS that kainate and L-glutamate cause fast onset and rapidly desensitizing responses in DRG neurones. These receptors displayed two predominant single-channel conductance levels of ~4 pS and 8 pS with infrequent openings of 15–18 pS (Ref. 39). Domoate and (S)-5-iodowillardiine, also elicit rapidly desensitizing responses in DRG (Refs 48, 54). This desensitizing effect of kainate in the DRG differs from its actions on AMPA receptors where nondesensitizing currents are observed. Despite reports of low kainate receptor subunit gene expression in the spinal cord, a population of cells express GluR5 and GluR7 and some cells contain KA2 mRNA within the DRG (Ref. 66). Interestingly, the pharmacological and desensitization profile of homomeric GluR5 receptors11,54 closely match those of the native DRG kainate recep32

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tors39,48,67. LY293558, which selectively antagonizes effects at homomeric GluR5, but not GluR6, also antagonizes the effects of kainate on DRG neurones52 and the multiple single-channel conductance levels of DRG kainate receptors are very similar to those of GluR5(Q) either expressed homomerically or in heteromeric combination with KA2 (Ref. 25). However, recent analysis of the desensitizing responses to (S)-5-iodowillardiine of kainate receptors has shown that homomeric GluR5 receptors show similar time course of recovery54 to native kainate receptors in the DRG (Ref. 48) whereas heteromeric GluR5/KA2 channels display a much faster recovery period54. Thus, although GluR5, GluR6 and KA2 are found in the DRG, it appears that the majority of native kainate receptors in this region are composed of homomeric assemblies of GluR5(Q). In contrast, investigations of kainate receptormediated responses in cultured trigeminal neurones have suggested that these receptors closely match the pharmacological and functional properties of recombinant receptors containing a heteromeric assembly of GluR5(R) and KA2 (Ref. 47).

CNS In addition to non-desensitizing responses mediated via AMPA receptors, the identification of rapidly desensitizing responses to kainate, domoate, L-glutamate and SYM2081, but not AMPA, in cultured hippocampal neurones have demonstrated the presence of native kainate receptors41,43,45,56. The selective AMPA receptor antagonist, GYKI53655, unmasks a small desensitizing response to kainate43. This desensitizing kainate-induced response was significantly inhibited by NS-102 and only partially antagonized by CNQX at concentrations that inhibit the non-desensitizing, AMPA receptor-mediated responses. Furthermore, the responses were not affected by GYKI52466, GYKI53655 or aniracetam43,45. As mentioned previously, NS-102 inhibits currents elicited at homomeric GluR6 receptors59 and single-cell reverse transcriptase polymerase chain reaction (rt–PCR) has shown that only GluR6 subunit mRNA is present in most of the cultured embryonic hippocampal neurones exhibiting the characteristic native kainate response30. Functional kainate receptors have also been identified on glial cell membranes and they display similar desensitization characteristics to those of neuronal kainate receptors68–70. It has been suggested, again using rt–PCR, that these receptors might comprise heteromeric assemblies of GluR6 and KA2 subunits69. Similarly, it has been shown that cultured cerebellar granule cells contain functional kainate receptors that resemble recombinant GluR6(R)/KA2 receptors; however, the presence of heteromeric GluR5/KA2 receptors cannot be ruled out51.

Synaptic transmission Because of the problems in identifying pure kainate receptor-mediated channel activity in the CNS, the physiological roles for kainate receptors have proved difficult to investigate. However, recent reports have

R revealed that kainate receptors can be activated by high-frequency electrical stimulation (for example 20 shocks at 100 Hz) of the mossy fibre but not of the associational/commissural pathway in hippocampus71,72. It has been proposed that the requirement for tetanic stimulation to elicit detectable kainate receptor-mediated currents implies substantial spillover out of the synaptic cleft of glutamate, which suggests an extrasynaptic location73. However, the fact that uptake block has no effect on the postsynaptic currents mediated by kainate receptors in the mossy fibre pathway is inconsistent with this possibility71,72. The subunit composition of the kainate receptors involved in mossy fibre synaptic transmission has been investigated using two selective antagonists for GluR5, namely LY293558 and LY294486. Both compounds reversibly antagonize kainate-induced currents in hippocampal CA3 neurones and also inhibit the synaptic activation of kainate receptors by a brief tetanus to the mossy fibre pathway74. Thus, kainate receptors containing or comprising the GluR5 subunit contribute to synaptic transmission in the hippocampus. However, the absence of kainate receptor-mediated excitatory postsynaptic currents in the CA3 region of GluR6 knockout mice75 suggests that GluR6-containing receptors are necessary for high-frequency synaptic transmission in this region. Therefore, at present, it still remains unclear as to the subunit composition of the postsynaptic kainate receptors involved in the excitatory postsynaptic currents observed in the CA3 region following stimulation of the mossy fibre pathway of the hippocampus.

Presynaptic kainate receptors In contrast to the now solid evidence for postsynaptic kainate receptors, there are contradictory results regarding presynaptic kainate receptors. Historically, the cellular localization of kainate receptors was initially investigated by lesioning studies. High-affinity [3H]kainate binding in CA3 region of hippocampus is significantly reduced following selective destruction of afferent mossy fibres, which suggests a presynaptic localization76,77. In mice that lack cerebellar granule cells, a decrease in [3H]kainate binding in the molecular layer (where the granule cell axons terminate) is observed compared to control animals78. Consistent with this presynaptic locus, immunostaining with an anti-KA2 subunit antibody stains mossy presynaptic terminals of the cerebellar granule layer64. In the same study, however, no GluR6/GluR7 antibody staining was observed in presynaptic terminals. Furthermore, GluR5/6/7 immunoreactivity was not observed in presynaptic terminals in monkey neocortex63. Nonetheless, the detection of kainate receptor immunoreactivity in unmyelinated axons could be suggestive of presynaptic receptors64,65. The neurotoxic effects of kainate in the hippocampus and striatum are attenuated when excitatory afferent projections are destroyed79,80. The fact that protection does not occur immediately after deafferentation but

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coincides with degeneration of nerve terminals led to the proposal that the neurotoxic action of kainate originates at a presynaptic locus81. Significantly, striatal cultures that are not susceptible to kainate-induced toxicity alone become sensitive when co-cultured with cortical neurones, suggesting the involvement of cortico–striatal afferents82. Another early study reported that kainate caused a Ca21dependent, tetrodotoxin-insensitive increase in endogenous glutamate and aspartate release from cerebellar slices comparable to that seen with KCl depolarization. This increase in release was prevented by afferent fibre degeneration83,84 and, on the basis of these data it was proposed that the action of kainate at presynaptic sites in glutamatergic synapses could explain its potent neurotoxicity. It has been observed that, rather than presynaptic kainate receptors having an excitatory action, kainate elicits a depolarization of isolated dorsal root fibres and a depression of certain electrically evoked C-fibre responses67,85. Recently, it has been shown that, unlike AMPA which stimulates, kainate elicits a concentrationdependent decrease in [3H]L-glutamate release from rat hippocampal synaptosomes and depresses glutamatemediated synaptic transmission in young rats86. Brief exposure to kainate inhibited Ca21-dependent [3H]L-glutamate release by up to 80%. Inhibition was reversed by CNQX and NS-102 but not by the AMPA receptorselective antagonist GYKI52466. Using synaptic activation of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) to provide a real-time monitor of glutamate release, a corresponding reversible kainateevoked depression in synaptically activated NMDA receptor-mediated EPSCs was observed when AMPA receptors were blocked by GYKI52466. Activation of kainate receptors has also been reported to downregulate GABA-mediated transmission in hippocampal CA1 neurones. Using a variety of analyses, this effect was attributed to a presynaptic action of kainate44,87,88. Taken together, these observations suggest that glutamate and GABA release is modulated by presynaptic kainate receptors, which serve as negative-feedback regulators. The combination of these inhibitory and disinhibitory presynaptic effects, together with the postsynaptic excitatory actions, could provide important mechanisms for the control of neuronal excitability.

Presynaptic role of GluR5 Characterization of presynaptic kainate receptors within the hippocampus has been further advanced by the GluR5 receptor-selective agonist ATPA and the selective antagonist LY294486. Using these compounds it was shown that kainate receptors comprising or containing GluR5 regulate synaptic inhibition by reducing evoked GABA release44. Kainate or ATPA, in the presence of GYKI53655 to block AMPA receptors, caused a reversible depression in inhibitory postsynaptic potentials (IPSPs). This depression of IPSPs was blocked by LY294486 (Ref. 44). Interestingly, it has been recently shown that this effect can also be prevented by pertussis

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toxin, which suggests that the mechanism of presynaptic inhibition occurs via a G protein-coupled cellular cascade89. In fact, this is the first functional evidence to suggest a metabotropic action of a population of kainate receptors. Because reduced monosynaptic GABA-mediated inhibition has been proposed as a mechanism underlying the epileptogenic actions of kainate87,90, kainate receptors might play an important role in the aetiology of epilepsy. Thus, GluR5-containing receptors could prove a useful target for the development of effective and selective antiepileptic drugs. However, a possible caveat of this approach that needs to be investigated in more detail is whether the presynaptic kainate receptors that downregulate excitatory glutamate-mediated synaptic transmission have the same subunit composition as the kainate receptors that inhibit GABA release.

Concluding remarks The use of recently developed AMPA and kainate receptor-selective agonists and antagonists has provided compelling evidence for functional pre- and postsynaptic kainate receptors in the mammalian CNS. These advances have added new impetus to the interest in kainate receptors. It is clear that subunit composition of kainate receptors influences their pharmacological and functional profiles. Recently, a number of studies have alluded to the subunit composition of these native receptors. The continuing development of drugs that not only selectively act at kainate receptors, but can also distinguish between kainate receptor subunits, coupled to the use of increasingly sophisticated and sensitive experimental techniques, such as rt–PCR, will increase our knowledge of the diversity of kainate receptors and lead to a fuller understanding of their functional roles.

Note added in proof The presynaptic kainate receptor-mediated inhibition of excitatory transmission in the CA1 region of the hippocampus86 has been extended to excitatory synapses in the CA3 region91. This effect can be elicited by ATPA and antagonized by LY294486 (Ref. 91). Thus, as with the postsynaptic kainate receptor-mediated currents found on CA1 pyramidal neurones74 and the presynaptic kainate receptors involved in the inhibition of inhibitory transmission in CA1 (Ref. 44), the kainate receptors involved in reducing excitatory transmission also contain or comprise GluR5 subunits. The downregulation of evoked GABA-mediated synaptic transmission in the CA1 region by kainate receptors has recently been confirmed92,93. Kainate receptor activation has been shown to cause a slight decrease in mini-IPSC frequency88,93 (but see Ref. 92) and this could account for the observed depression of evoked IPSCs in CA1 pyramidal neurones. However, the recently described kainate receptor-mediated depolarization of interneurones and consequent increase in action potential discharge causing the spontaneous release of GABA might underlie the depression of evoked GABA-mediated transmission by kainate 34

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receptor activation92,93. Finally, afferent activation also causes a kainate receptor-mediated postsynaptic current in interneurones92,93, which is sensitive to the nonselective AMPA and kainate receptor antagonist LY293558 (Ref. 93) and has distinct kinetic properties to AMPA receptor-mediated currents92,93. Thus, recently published data have served to confirm the existence of functional kainate receptors in the CNS and give further evidence for the role of the GluR5 subunit in determining the excitability of the hippocampal neuronal circuitry. References 1 Watkins, J. C. and Evans, R. H. (1981) Annu. Rev. Pharmacol. Toxicol. 21, 165–204 2 Bettler, B. et al. (1990) Neuron 5, 583–595 3 Herb, A. et al. (1992) Neuron 8, 775–785 4 Werner, P. et al. (1991) Nature 351, 742–744 5 Bettler, B. et al. (1992) Neuron 8, 257–265 6 Egebjerg, J. et al. (1991) Nature 351, 745–748 7 Lomeli, H. et al. (1992) FEBS Lett. 307, 139–143 8 Bettler, B. and Mulle, C. (1995) Neuropharmacology 34, 123–139 9 Hollmann, M. and Heinemann, S. (1994) Annu. Rev. Neurosci. 17, 31–108 10 Wo, Z. G. and Oswald, R. E. (1995) Trends Neurosci. 18, 161–168 11 Sommer, B. et al. (1992) EMBO J. 11, 1651–1656 12 Schiffer, H. H., Swanson, G. T. and Heinemann, S. F. (1997) Neuron 19, 1141–1146 13 Gregor, P. et al. (1993) NeuroReport 4, 1343–1346 14 Sommer, B. et al. (1991) Cell 67, 11–19 15 Bernard, A. and Khrestchatisky, M. (1994) J. Neurochem. 62, 2057–2060 16 Paschen, W., Dux, E. and Djuricic, B. (1994) Neurosci. Lett. 174, 109–112 17 Paschen, W. et al. (1995) Dev. Brain Res. 86, 359–363 18 Schmitt, J. et al. (1996) Dev. Brain Res. 91, 153–157 19 Köhler, M. et al. (1993) Neuron 19, 491–500 20 Swanson, G. T., Kamboj, S. K. and Cull-Candy, S. G. (1997) J. Neurosci. 17, 58–69 21 Sakimura, K. et al. (1992) Neuron 8, 267–274 22 Howe, J. R. (1996) J. Neurophysiol. 76, 510–519 23 Egebjerg, J. and Heinemann, S. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 755–759 24 Burnashev, N. et al. (1995) J. Physiol. 485, 403–418 25 Swanson, G. T. et al. (1996) J. Physiol. 492, 129–142 26 Burnashev, N., Villarroel, A. and Sakmann, B. (1996) J. Physiol. 496, 165–173 27 Burnashev, N. (1996) Curr. Opin. Neurobiol. 6, 311–317 28 Kamboj, S. K., Swanson, G. T. and Cull-Candy, S. G. (1995) J. Physiol. 486, 297–303 29 Bowie, D. and Mayer, M. L. (1995) Neuron 15, 453–462 30 Ruano, D. et al. (1995) Neuron 14, 1009–1017 31 Honoré, T. et al. (1988) Science 241, 701–703 32 Sheardown, M. J. et al. (1990) Science 247, 571–574 33 Lodge, D., Jones, M. G. and Palmer, A. J. (1991) Can. J. Physiol. Pharmacol. 69, 1123–1128 34 Bleakman, D. et al. (1996) Neuropharmacology 35, 1689–1702 35 Wilding, T. J. and Huettner, J. E. (1996) Mol. Pharmacol. 49, 540–546 36 Paternain, A. V. et al. (1996) Eur. J. Neurosci. 8, 2129–2136 37 Partin, K. M. et al. (1993) Neuron 11, 1069–1082 38 Wong, L. A. and Mayer, M. L. (1993) Mol. Pharmacol. 44, 504–510 39 Huettner, J. E. (1990) Neuron 5, 255–266 40 Everts, I., Villmann, C. and Hollmann, M. (1997) Mol. Pharmacol. 52, 861–873 41 Wilding, T. J. and Huettner, J. E. (1995) Mol. Pharmacol. 47, 582–587 42 Ouardouz, M. and Durand, J. (1991) Neurosci. Lett. 125, 5–8 43 Paternain, A. V., Morales, M. and Lerma, J. (1995) Neuron 14, 185–189 44 Clarke, V. R. J. et al. (1997) Nature 389, 599–603 45 Lerma, J. et al. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11688–11692 46 Lauridsen, J., Honoré, T. and Krogsgaard-Larsen, P. (1985) J. Med. Chem. 28, 668–672 47 Sahara, Y. et al. (1998) J. Neurosci. 17, 6611–6620 48 Wong, L. A. et al. (1994) J. Neurosci. 14, 3881–3897 49 Jones, K. A. et al. (1997) Neuropharmacology 36, 853–863 50 Zhou, L. M. et al. (1997) J. Pharmacol. Exp. Ther. 280, 422–427 51 Pemberton, K. E. et al. (1998) J. Physiol. 510, 401–420 52 Bleakman, D. et al. (1996) Mol. Pharmacol. 49, 581–585 53 Hawkins, L. M. et al. (1995) Br. J. Pharmacol. 116, 2033–2039

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The pharmacology of apoptosis Ross A. Kinloch, J. Mark Treherne, L. Mike Furness and Iradj Hajimohamadreza Apoptosis is an area of intense scientific interest, which encompasses the study of and triggers mechanisms involved in mediating the cell biology of programmed cell death. A number of low molecular weight compounds have been used to inhibit or enhance this fundamental cellular process and so apoptosis has now become amenable to pharmacological manipulation. In this review Ross Kinloch, Mark Treherne, Mike Furness and Iradj Hajimohamadreza will focus on the current literature describing the pharmacology of apoptosis, with particular reference to the therapeutic potential that could arise from the development of proand anti-apoptotic drugs. The pivotal role of apoptosis in such diverse pathological processes as tumour growth, the immune response and neurodegeneration suggests that an understanding of how apoptosis can be regulated by drugs will become increasingly important to the pharmaceutical industry.

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Chemical names GYKI52466: 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine GYKI53655; LY300168: 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydroxy5H-2,3-benzodiazepine LY293558: (3S, 4aR, 6R, 8aR)-6-[2-(1(2)H-tetrazol-5-yl)ethyl] decahydroisoquinoline-3-carboxylic acid LY294486: (3SR, 4aRS, 6SR, 8aRS)-6-((((1H-tetrazol-5yl)methyl)oxy)methyl)-1,2,3,4,4a,5,6,7,8,8a decahydroisoquinoline-3-carboxylic acid SYM2081: (2S,4R)-4-methylglutamic acid

Apoptosis describes the complex contortions of the cell membrane and organelles of a cell as it activates an intrinsic suicide programme and systematically destroys itself. Although the term ‘apoptosis’ was first used in this context almost 25 years ago, it is only in the past five years that research into this phenomenon has really taken off in terms of understanding the underlying mechanisms. The rapid increase in publications in the field of apoptosis reflects a transition from descriptive cell biology to detailed studies on the mechanisms driving the machinery of cell death. There have been a number of good reviews on the subject1–9 but none appear to have focused on how low-molecular-weight molecules have been used to inhibit or enhance this process. Therefore, particular reference will be paid to the therapeutic potential that will come from the application of pharmacology to the cell biology of programmed cell death. Perhaps the most obvious physiological role of apoptosis is that which occurs during development, but this fundamental process of cell biology also occurs during the normal turnover of adult tissues as well as during pathological conditions. Interfering with developmental apoptosis, however, would appear to confer little obvious therapeutic benefit, but pharmacologists should be aware of the potential restriction in the use of pro- and anti-apoptotic drugs during pregnancy. Apoptosis plays an important role in the turnover of rapidly dividing tissue (such as intestinal villi and lymphocytes) and a decrease in the rate of apoptosis might have severe pathological consequences and might facilitate the growth of tumours. Cell death occurs normally in the liver but this

0165-6147/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S0165-6147(98)01277-2

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Acknowledgements We would like to thank the BBSRC, Eli Lilly, MRC and the Wellcome Trust for financial support.

R. A. Kinloch, Senior Principal Scientist, Department of Discovery Biology, Pfizer Central Research, Sandwich, UK CT13 9NJ, J. M. Treherne, Chief Executive Officer, Cambridge Drug Discovery, Science Park, Milton Road, Cambridge, UK CB4 4FD, L. M. Furness, Project Leader, Incyte Europe, Botanic House, 100 Hills Road, Cambridge, UK CB2 1FE, and I. Hajimohamadreza, Senior Scientist, Department of Discovery Biology, Pfizer Central Research, Sandwich, UK CT13 9NJ.

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