WO2004056867A2

WO2004056867A2 - Ion channel for testing pharmacological active compounds

Info

Publication number: WO2004056867A2
Application number: PCT/GB2003/005502
Authority: WO
Inventors: Lucia Sivilotti; Paul Groot-Kormelink
Original assignee: University College London
Current assignee: University College London
Priority date: 2002-12-19
Filing date: 2003-12-18
Publication date: 2004-07-08
Anticipated expiration: 2005-06-19
Also published as: WO2004056867A3; AU2003295126A8; AU2003295126A1; GB0229586D0

Abstract

This invention relates to ligand-gated ion channels in the nicotinic superfamily and methods for constraining their subunit composition. This invention provides a polypeptide chain comprising four or five of the subunits of a ligand-gated ion channel. This polypeptide chain is able to form a functional ligand-gated ion channel in combination with another subunit or on its own respectively.

Description

NOVEL TOOL FOR TESTING PHARMACOLOGICAL ACTIVES

This invention relates to ligand-gated ion channels in the nicotinic superfamily also called neurotransmitter receptors and methods for constraining their subunit composition.

Ligand-gated ion channels or receptors in the nicotinic superfamily (also called nicotinicoid superfamily) are each composed of five subunits. Each subunit comprises an N-terminal extracellular domain, which is involved in binding to a ligand eg a neurotransmitter, four hydrophobic transmembrane domains, a long cytoplasmic loop between the third and fourth transmembrane domains and shorter loops connecting the remaining transmembrane domains. Binding of a specific ligand to the ligand-gated ion channel induces a conformational change opening the channel allowing ions to pass through. Ligand-gated ion channels are generally found at synapses and mediate transmission of a chemical signal to an electrical signal in the subsequent neurone. These ligand-gated ion channels are linked to neurodegenerative disorders such as Alzheimer's, Parkinson's, schizophrenia and epilepsy, and mediate tobacco addiction. There is much research centred on these ligand-gated ion channels, and the neurotransmitters and other actives which modulate the effects of ligand- gated ion channels.

Pharmacological research is hampered by the complexity shown by these ligand-gated ion channels. For example the neuronal nicotinic receptors are generally composed of two α and three β subunits. So far nine different α subunits have been identified (α2, α3, α4, α5, α6, α7, αδ, α9 and α10) and three β subunits (β2, β3 and β4). GABA receptors are believed to be composed of subunits from six classes each with several isoforms (α1-6, β1-3, Y 1-3, ε, δ, θ, π). ( Le Novere et al, Inc. J Neurobiol (2002) 53:447-456) The large number of different subunits that can be expressed in vivo makes it difficult to know the exact make-up of the receptors present in neurones. Functional recombinant receptors have been produced from constructs of these genes expressed in heterologous systems in vitro to obtain receptors that can be used for all sorts of biophysical, biochemical and pharmacological studies. Neuronal nicotinic receptors can be produced by expressing one subunit only (if the subunit is α7 or α9), by expressing an α and a β subunit together (α2-α4 plus β2 or β4) or in a triplet fashion by expressing an α together with β and either α5 or β3.

While these recombinant receptors are functional, there is no guarantee that they do reproduce the native receptors (e.g present in human brain and autonomic ganglia). This is because neurones do not as a rule express just the one or two subunits that are sufficient to form a receptor. The formation of receptors containing three or even four different subunits has been demonstrated by immunoprecipitation techniques (Conray & Berg, J.Biol.Chem., 270(1995), 4424; Forsayeth & Kobrin, J.Neurosci., 17(1997(, 1531). However, not only do such techniques rely on developing subunit- specific antibodies, but once the existence of complex heteromeric receptors is confirmed, immunoprecipitation does not provide further information in terms of characterization of the receptor in a pharmacological sense, as - at best- only binding assays might be feasible on the immunoisolated receptor. A much more attractive proposition is the expression of subunits in an in vitro heterologous system. Nevertheless, this route is not practicable, because injecting mixes of constructs, each coding for a single subunit results in a mixed heterogeneous population consisting of a mixture of several receptor types. This mixture is useless for the screening of pharmacological agents. Previous studies in this area have produced three main attempts at restricting subunit composition: all were carried out on GABA_A receptors and involved the manufacture of dimer constructs, containing the coding regions for two subunits (Im et al., J. Biol. Chem.270 (1995), 26073; Baumann et al., J.Biol. Chem. 276 (2001 ), 36275) or a dimer combined with a trimer construct (Baumann et al., J. Biol. Chem. 277(2002) pp 46020-46025. The dimer constructs are expressed together with a monomer, or a dimer with a trimer in the latest study. It was believed that receptors produced in this way were functional and mimicked natural receptors. The inventors of the current invention however, have shown this not to be the case. In studies aimed at restricting the stoichiometry of recombinant nicotinic acetylcholine receptors (nAChRs), dimer constructs were created by linking polynucleotides encoding α3 and β4 subunits via short linker regions and thesewere expressed in Xenopus oocytes along with different monomer subunits. Oocytes were then tested with acetylcholine, using two-electrode voltage clamp, to see which tandem/monomer combinations produced functional receptors. Dimers of the α3-β4 orientation failed to produce functional receptors either when expressed alone or along with any monomer. Dimers of the β4- α3 orientation did not produce functional receptors when expressed alone or along with α3, α4, α6 or β3 subunits (small responses were observed with monomer α2, α5 or β2 subunits). When the β4-α3 tandem was expressed along with β4 subunits, large inward currents were observed in response to acetylcholine. Dose-response curves were therefore obtained for β4-α3 + β4 receptors (n=4) (wherein n is the number of independent experiments) and compared with those from α3β4 receptors expressed from monomeric constructs (n=10). The tandem-containing receptors were found to be macroscopically indistinguishable from α3β4 receptors (EC₅₀=125 + 13 versus. 137+24 μM: n_H (Hill Coefficient) = 1.98 + 0.47 versus. 1.76 + 0.16, means + S.D.). Therefore a functional receptor had been obtained.

However, the inventors wanted to check that receptors expressed from polynucleotide constructs encoding β4-α3 with β4 did actually contain two copies each of the α and β subunits from the tandem construct and one copy of the subunit from the β monomer cRNA. This was done by mutating from leucine to threonine the 9^th residue in the pore-lining region of the β subunit contained in the tandem construct. In channels of the nicotinic superfamily, incorporation of this mutation (β4^LT) increases the receptor agonist sensitivity by an amount that is roughly proportional to the number of mutation copies present. Expressing β4 - α3 + β4 ^T should give rise to a homogeneous population of receptors with a dose-response curved shifted to the left because of the presence of a single copy of the mutation. Nevertheless, dose-response curves obtained from such mutant combinations typically showed more than one component, i.e could not be described by a single Hill equation with a single EC₅₀ but requires at least two Hill equations with different EC₅₀. This indicates the simultaneous presence in the receptor population of at least two populations of channels containing different numbers of mutation copies and therefore different numbers of β subunit produced by the monomer cRNA.

This means that the receptor expressed from dimers with monomer (βα + β) did not have the composition expected. The expected composition is two α subunits from the dimers and three β subunits, two from the dimers and one from a monomer. Indeed mutation of subunits in the dimer expression experiments shows that the receptors are likely to be a mixed population probably because some of the receptors contain "trailing subunits", that is incorporate only one of the subunits in the dimer construct. Our reporter mutation data therefore indicate that the dimer construct approach is useless because the dimer can act as a proper dimer, but also as a monomer (with a large "tag") and therefore still does not produce a single population of expressed receptors with fixed stoichiometry/topology. The inventors' experiments demonstrated the existence of this problem which was not previously appreciated and they developed the product of the present invention to alleviate the problems of the prior art. The present invention is a polypeptide chain which comprises four subunits of a ligand-gated ion channel and is able form a functional ligand- gated ion channel in combination with another subunit. This is a method by which the subunit composition of ligand-gated ion channels can be constrained and prevent the presence of "trailing subunits".

In an alternative embodiment the present invention is a polypeptide chain which comprises five subunits of a ligand-gated ion channel and is able to form a functional ligand-gated ion channel. This invention is applicable to receptors across the large superfamily of ligand-gated ion channels. This invention is applicable to the cys-loop receptor superfamily and to the nicotinicoid superfamily of receptors. (Le Nouvere et al Nucleic Acids Research (2001) 29:1 , pp 294-295). This includes channels which respond to any one of acetylcholine (ACh), γ-amino butyric acid (GABA), glycine, 5HT(serotonin) and nicotine.

The ability to constrain subunit composition will facilitate studies investigating the subunit composition of natural ligand-gated ion channels. The processes of the present invention can be used for any type of ligand-gated ion channel formed from five subunits. GABA receptors, for example, may be formed from six different types of subunits each with several isoforms. However, the preferred embodiment of this invention is focussed on nicotinic acetylcholine receptors formed from α and β subunits. The ligand-gated ion channel database LGICdb

(http://www.pasteur.fr/recherche/banques/LGIC/LGIC.html) is a repository of information relating to ligand-gated ion channels and their subunits. This invention is especially relevant to the superfamily of nicotinicoid receptors (nicotinic receptors, GABA_A and GABA_C receptors, glycine receptors, 5-HT₃ receptors and some glutamate activated anionic channels) and mutated variants thereof able to form functional channels. (The LGICdb provides an interface to programs capable of screening sequences and indicating similarities between them).

Subunits designated as α have a pair of adjacent cysteine residues located at the N-terminal region at positions corresponding to residue 192 and 193 in the mature Torpedo α1 subunit. Subunits lacking the pair of adjacent cysteine residues are non- α subunits, for example β subunits.

Sequence number 1 , α3 subunit and sequence ID Nos. 1 and 2 provide the DNA and protein sequence of an α3 subunit. α subunits which may be used in the methods of this invention have 50%, 60%, 70%, 80%, 90%, 95% similarity or identity with this sequence and can be used to form a functional ligand-gated ion channel. Sequence number 2, β4 subunit and sequence ID Nos. 3 and 4 provide the DNA and protein sequence of a β4 subunit. β subunits which may be used in the methods of this invention have 50%, 60%, 70%, 80%, 90%, 95% similarity or identity with this sequence and can be used to form a functional ligand-gated ion channel. Generally such channels are formed from two α and three β subunits. The terms as used above, "similarity" and "identity" are known in the art. The use of the term "identity" refers to a sequence comparison based on identical matches between correspondingly indentical positions in the sequences being compared. The term "similarity" refers to a comparison between amino acid sequences, and takes into account not only identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, in addition to sequence similarity. Levels of identity between gene sequences and levels of identity or similarity between amino acid sequences can be calculated using known methods. In relation to the present invention, publicly available computer based methods for determining identity and similarity include the BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol., 1990; 215:403-410), the BLASTX program available from NCBI, and the Gap program from Genetics Computer Group, Madison Wl.

This invention relates to ligand-gated ion channels and their subunits which have recorded sequences and mutated versions of subunits capable of binding a ligand and forming part of a ligand-gated ion channel. The subunit function can be tested by expressing the subunit on its own and with other subunits in turn in a heterologous system to find a functional combination. An example of such an expression method is as described in Examples 2 and 3 below.

Preferably a channel is formed from two identical α subunits and three identical β subunits. More preferably the channel is formed from α3 and β4 subunits.

In the most preferred version the channel is formed from subunits which are connected in the order β4-β4-α3-β4-α3.

The subunits found in ligand-gated ion channels according to the present invention may be connected via linker regions. Such linker regions can vary widely in their length and composition. For example linker regions must be of sufficient length to allow two consecutive subunits to orient themselves suitably for the formation of the ligand-gated ion channel. Excessively long linker regions could interfere with the formation of the ligand-gated ion channel. It is preferred that the coding sequences for the linker regions comprise restriction enzymes sites. These are useful in manipulation of sequences, necessary to form the desired constructs. The linker regions may all be identical. Alternatively each linker may differ from the others for example the coding sequence for each linker having a unique restriction site.

The ligand-gated ion channel of the present invention is generally manipulated in the form of a polynucleotide sequence. Therefore a second aspect of this invention is a polynucleotide encoding a polypeptide of the present invention. A third aspect is a polynucleotide in a form suitable for expression in a cell. The polynucleotide in the form of a capped mRNA molecule would be a suitable form for expression in certain cells for example oocytes. Whereas a vector comprising the polynucleotide would be a form suitable for transfections of other cells. As described above research is carried out on determining the types of ligand-gated ion channels present in different neurones and various pharmacological tests are dependent upon obtaining a homogeneous population of ligand-gated ion channels. Therefore a fourth aspect of this invention provides a method for obtaining a homogeneous population of ligand-gated ion channels comprising expressing a polynucleotide encoding a ligand-gated ion channel of the present invention. Usually the polynucleotide would be expressed in a heterologous system. Preferably the polynucleotide is expressed in a cell which does not normally produce ligand- gated ion channels. More preferably the polynucleotide is expressed in oocytes. Most preferably the polynucleotide is expressed in Xenopus oocytes, for example an immature oocyte.

As described above much research is currently focussed on obtaining pharmaceuticals which can prevent and/or alleviate the symptoms of neurodegenerative disorders. A fifth aspect is a method for assaying actives of such type for potential activity comprising; a) producing a homogeneous population of ligand-gated ion channels according to the present invention, b) bringing the ligand-gated ion channels into contact with a ligand to generate a response, c) bringing the ligand-gated ion channels into contact with an active, d) detecting and measuring any electrical response which is generated across the ligand-gated ion channel.

Binding of a ligand to a ligand-gated ion channel can cause a conformational change to occur in the transmembrane domains of the channel which line the pore. The conformational change can open the pore allowing a flow of ions across the membrane in which the ligand-gated ion channel is situated.

The electrical response can be measured when the membrane potential of the cell is clamped to a fixed, physiological value. The electrical response consists of a current which corresponds to the flow of ions through the open channels and can be measured by the voltage-clamp amplifier. This is measured by measuring how much current is needed to keep the cell at a fixed potential.

In some instances the ligand to which the ligand-gated ion channel responds and the active are the same substance.

In the method for assaying actives it is envisioned that actives may be agonists (which activate the channel), antagonists (which close the channel), blockers (which physically block the pore to close the channel) and modulators (which can enhance or reduce the effects of other ligands).

Preferably, the step of producing a homogeneous population of ligand-gated ion channels according to the present invention comprises expressing a polynucleotide encoding a ligand-gated ion channel according to the present invention.

The inventors of the present invention have also identified an additional problem with the prior art, that its current techniques do not allow for the expression of neuronal nicotinic or GABA receptors which contain one and only one copy of the mutation in subunits present in more than copy. This poses significant limitations on the study of receptor activation particularly in connection with human channelopathies. These inherited diseases are commonly inherited in a dominant pattern in that the patient has one mutant and one normal allele (see for instance autosomal dominant nocturnal frontal lobe epilepsy for neuronal nicotinic receptors and hyperekplexia for glycine receptors). In individuals such diseases ligand- gated ion channels will be formed comprising subunits produced from the normal and the mutant alleles. Therefore a channel may contain one or two normal versions of a subunit, but still show a disease phenotype because a single mutant subunit is present. Current techniques for production of recombinant channels based on monomers and dimers will allow incorporation of mutant subunits, but each subunit of a particular type will posses the mutation rather than only one subunit of a particular type as potentially found in the disease state. Once all five subunits are present in a continuous polypeptide or encoded by one polynucleotide sequence it is possible to introduce a mutation in one copy of the subunit appearing in a plurality of copies. Therefore a sixth aspect is a method for introducing a mutation into one copy of a subunit appearing in a ligand-gated ions channel in a plurality of copies, comprising introducing the mutation into a polynucleotide encoding the ligand-gated ion channel of the present invention.

This invention will be discussed further below in relation to Figures 1 to 10 wherein;

Figure 1 shows the topology of a single subunit and a subunit dimer, Figure 2 shows the arrangement of subunits in a ligand -gated ion channel, Figure 3 shows creation of α3-β4 dimer construct, Figure 4 shows creation of β4-α3 dimer construct, Figure 5 shows subcloning of α3-β4 dimer construct into an alternative plasmid,

Figure 6 shows subcloning of β4-α3 dimer construct into an alternative plasmid, Figure 7 shows further subcloning of α3-β4 dimer construct into another plasmid,

Figure 8 shows further subcloning of β4-α3 dimer construct into a different plasmid,

Figure 9 shows creation of β4-β4-α3 trimer construct, Figure 10, shows creation of β4-β4-α3-β4-α3 pentamer construct.

Figure 1A shows the transmembrane topology of a single subunit in the ligand-gated ion channel superfamily. Figure 1 B shows two subunits connected by a linker. This construct arrangement is part of the prior art. Figure 2A shows, schematically, the pentameric ligand-gated ion channel formed from five monomeric subunit constructs. This is the arrangement in vivo. Figure 2B schematically illustrates the pentameric ligand-gated ion channel of the present invention formed by five subunits linked together in a single continuous polypeptide. This invention will be further illustrated by reference to the following examples.

Example 1

This example describes a method to obtain a ligand-gated ion channel according to the present invention. 1) Subcloning of the human α3 and β4 cDNAs. cDNAs for the human neuronal nicotinic acetylcholine receptor subunits α3 and β4 (GenBank accession numbers Y08418 and Y08416 respectively), containing only coding sequences (CDS) and an added Kozak consensus sequence (GCCACC) immediately upstream of the start codon (Groot-Kormelink & Luyten, 1997), were subcloned into two different vectors; pSP64GL (contains 5' and 3' untranslated Xenopus β-globin regions (Akopian et al., 1996) for oocyte expression studies) and pcDNA3.1 + (mammalian expression vector, Invitrogen, The Netherlands). These plasmid constructs will be referred to as α3/pSP64GL [1], β4/pSP64GL [2], α3/pcDNA3.1 [3], and β4/pcDNA3.1 [4] respectively. Both cDNAs were also cloned into the epitope-tagged vector pcDNA3.1_myc-His version C (Invitrogen, The Netherlands). In order to clone the insert in frame with the 3' epitope tags (myc and His followed by the stop codon) the stop codons of the α3 and β4 subunit cDNAs have to be removed. Therefore, the α3 and β4 CDS were amplified by standard PCR using PCR primers that amplified only the CDS region up to but excluding the stop codon. Furthermore, two restriction enzyme sites were introduced in the primers, in order to facilitate the subsequent cloning of the PCR product into the pcDNA3.1_myc-His version C vector. The final PCR products for both α3 and β4 are [EcoRI - GCCACCATG....CDS (α3 or (34)....Notl]. The EcoRI and Notl restriction enzyme sites were chosen because they do not digest any of the neuronal nicotinic acetylcholine receptor subunit cDNAs (this will allow us to extend the same strategy to the rest of the neuronal nicotinic acetylcholine receptor family). They also enable directional cloning into the pcDNA3.1_myc-His version C vector in the correct frame. These epitope tagged plasmid constructs are named α3/pcDNA3.1_myc-His [5] and β4/pcDNA3.1_myc-His [6], respectively.

Both constructs (5 and 6) were sequenced fully on both strands from the T7 promotor to the BGH pA sequence and were found to be 100% correct. 2) Construction of the β4_β4_a3_β4_a3 pentamer. In order to construct the pentamer several dimer and trimer constructs were made. All constructs (dimer, trimer, and pentamer) were linked in an identical fashion using the same linker. 2a) Construction of the linker A double stranded linker was made by adding two complementary oligonucleotides (0.5 mg/ml final concentration) to restriction enzyme reaction buffer M (Roche, UK), heating them to 95 °C for 5 minutes followed by cooling down slowly to room temperature (i.e. in the switched off heating block) and storing them at -20 °C. The following oligonucleotides were used in this study; 5' Primer: GGCCGCTCAGCAACAGCAGCAACAGCAGCAAG 3' Primer: AATTCTTGCTGCTGTTGCTGCTGTTGCTGAGC The resulting linker will have 'sticky' ends of the Notl and EcoRI restriction enzyme sites that can be used to ligate them to DNA fragments digested with EcoRI and Notl. This approach was chosen because by ordering different oligonucleotides one can very easily change the restriction enzyme ends or the linking sequence. For instance to shorten or lengthen the linker, to introduce epitope tags for biochemical studies etc. 2b) Construction of the dimers

All the dimers were constructed identically by using the restriction enzyme sites EcoRI, Notl and Agel;

EcoRI sites; immediately upstream of the start codon of the α3 and β4 cDNAs in construct 5 and 6) and on the 3' end of the 'sticky' linker. Notl sites: instead of the stop codon in of the α3 and β4 cDNAs in construct 5 and 6 and on the 5' end of the 'sticky'.

Agel site: The Agel site is uniquely situated on the pcDNA3.1/tτ7yc-His version C vector between the epitope tag sequences of myc and His.

Note that there is no EcoRI, Notl, or Agel restriction enzyme site in any of the neuronal nicotinic cDNA sequences which makes the approach applicable for any combination of neuronal nicotinic dimers (see also below).

The dimer construct is created by a ligation reaction of three DNA fragments (see Figures 3 and 4). The resulting dimer constructs are named α3_β4/pcDNA3.1_myc-His [7] and β4_α3/pcDNA3.1_ yc-His [8]. In order to have dimer constructs without the epitope tags we subcloned the dimer constructs into the pcDNA3.1 vector (see Figures 5 and 6). The resulting dimer constructs are named α3_β4/pcDNA3.1 [9] and β4_α3/pcDNA3.1 [10]. Furthermore, we subcloned the tandem constructs into the pSP64GL vector (see Figures 7 and 8) resulting in α3_β4/pSP64GL [11] and β4_α3/pSP64GL [12]. Both constructs (11 and 12) were sequenced fully on both strands from the

T7 promotor to the BGH pA sequence and were found to be 100% correct.

2c) Construction of the β4_β4__a3 trimer

The trimer was constructed by ligation of three DNA fragments originating from the following digests; β4_α3/pSP64GL [12, digested with Xbal and

EcoRI], β4/pcDNA3.1_/??yc-His [6, digested with EcoRI/Apal], and β4_α3/pSP64GL [12, digested with Xbal and Apal]. See Figure 9. The resulting trimer was named β4_β4_α3/pSP64GL [13].

2d) Construction of the β4_β4_a3J34_a3 pentamer The pentamer was constructed by ligation of three DNA fragments originating from the following digests; β4_β4_α3/pSP64GL [13, digested with

Kspl and Xbal], a3_b4/pcDNA3.1_myc-His [7, digested with Kspl and Apal], β4_α3/pSP64GL [12, digested with Apal and Xbal]. See Figure 10. The resulting pentamer was named β4_β4_α3_β4_α3/pSP64GL [14]. The same approach can be used to make any pentameric construct, all with identical linkers.

Example 2

Capped mRNAs are required for expression of the pentameric construct in

Xenopus oocytes. Preparation of in vitro capped mRNAs

The following plasmid constructs were used to make in vitro-capped mRNAs; α3/pSP64GL [1], β4/pSP64GL [2], and β4_β4_α3_β4_α3/pSP64GL [14]. All three cDNA/pSP64GL plasmids were linearised immediately downstream of the 3' untranslated β-globin sequence, and in vitro capped RNA (cRNA) was transcribed using the SP6 Mmessage Mmachine Kit (Ambion). The quality and quantity was checked by RNA gel-electrophoresis and comparison with RNA concentration and size markers. Example 3 Testing the ligand-gated ion channels formed from constructs produced according to examples 1 and 2. 1)Xenopus oocyte preparation and electrophysiological recording

Female Xenopus laevis frogs were anaesthetised by immersion in neutralised ethyl m-aminobenzoate solution (tricaine, methanesulphonate salt; 0.2% solution weight/volume; Sigma Chemical Co.), and killed by decapitation and destruction of the brain and spinal cord (in accordance to Home Office guidelines) before removal of ovarian lobes. Clumps of stage V-VI oocytes were dissected in a sterile modified Barth's solution of composition (in mM): NaCI 88; KC1 1 ; MgCI₂ 0.82; CaCI₂ 0.77; NaHC0₃ 2.4; Tris-HCI 15; with 50 U ml^"1 penicillin and 50 mg ml^"1 streptomycin; pH 7.4 adjusted with NaOH. The dissected oocytes were treated with collagenase (type IA, Sigma Chemical Co.; 65 minutes at 18° C, 245 collagen digestion units ml ¹ in Barth's solution, 10-12 oocytes per ml), rinsed, stored at 4° C overnight, and manually defolliculated the following day before cRNA injection (46 nl per oocyte). The oocytes were incubated for approximately 60 hours at 18° C in Barth's solution containing 5% heat-inactivated horse serum (Gibco BRL; Quick and Lester, 1994) and then stored at 4° C. Experiments were carried out at a room temperature of 18-20° C between 2.5 and 14 days from injection. cRNA was injected at a ratio of 1 :1 in order to express receptors from the monomers α3 and β4s. The total amount of cRNA to be injected (in 46 nl of RNAse-free water) was determined empirically, with the aim of achieving a maximum ACh-evoked current of 1.5-2 mA. Oocytes, held in a 0.2 ml bath, were perfused at 4.5 ml/min with modified Ringer solution (NaCI 150, KCI 2.8, HEPES 10, MgCI₂2 mM, 0.5mM atropine sulphate, Sigma Chemical Co.; pH 7.2 adjusted with NaOH) and voltage clamped at -70 mV, using the two-electrode clamp mode of an Axoclamp-2B amplifier (Axon Instruments). Electrodes were pulled from Clark borosilicate glass GC150TF (Warner Instrument Corporation) and filled with 3 M KCI. The electrode resistance was 0.5-1 MΩ on the current-passing side. Experiments were terminated if the total holding current exceeded 2 μA, in order to reduce the effect of series resistance errors. We chose a nominally calcium-free solution in order to minimise the contribution of calcium-gated chloride conductance; this is endogenous to the Xenopus oocyte and may be activated by calcium entry through the neuronal nicotinic channels. The agonist solution (acetylcholine chloride, Sigma Chemical Co.; freshly prepared from frozen stock aliquots) was applied via the bath perfusion, for a period sufficient to obtain a stable plateau response (at low concentrations) or the beginning of a sag after a peak (at the higher concentrations); the resulting inward current was recorded on a flat bed chart recorder (Kipp & Zonen) for later analysis. An interval of 5 minutes was allowed between ACh applications, as this was found to be sufficient to ensure reproducible responses. In order to compensate for possible decreases in agonist sensitivity throughout the experiment, a standard concentration of ACh (approximately EC₂₀ for the particular combination used) was applied every third response. The experiment was started only after checking that this standard concentration gave reproducible responses. 2) Curve fitting

All dose-response curves were fitted with the Hill equation

where / is the response, measured at its peak, [A] is the agonist concentration, /_max is the maximum response, £C₅₀ is the agonist concentration for 50% maximum response and n_H is the Hill coefficient. We used least squares fitting by the program CVFIT, courtesy of D. Colquhoun and I. Vais, available from http://www.ucl.ac.uk/Pharmacology/dc.html.

Fitting was done in stages, as follows. Each dose-response curve was fitted separately, individual responses being equally weighted, in order to obtain estimates for l_max, EC₅₀ and n_H. The means and standard deviation of the means for each combination are shown in Table 1. Tablel ; EC₅₀ values were obtained by fitting separately the individual concentration-response curves.

This indicates that the ligand-gated ion channel formed from the pentameric construct is functionally identical to a channel formed from the corresponding individual subunits. Therefore this is a valid model for testing actives in ligand-gated ion channels.

Sequence Number 1 α3 subunit

M A ATGGCTCTGG TACCGAGACC

A V S P L A L S P P R L L L CCGTCTCGCT GCCCCTGGCG CTGTCGCCGC CGCGGCTGCT GCTGCTGCTG GGCAGAGCGA CGGGGACCGC GACAGCGGCG GCGCCGACGA CGACGACGAC

L S L L P V A R A S E A E H R L F CTGTCTCTGC TGCCAGTGGC CAGGGCCTCA GAGGCTGAGC ACCGTCTATT GACAGAGACG ACGGTCACCG GTCCCGGAGT CTCCGACTCG TGGCAGATAA

E R L F E D Y N E I I R P V A N TGAGCGGCTG TTTGAAGATT ACAATGAGAT CATCCGGCCT GTAGCCAACG ACTCGCCGAC AAACTTCTAA TGTTACTCTA GTAGGCCGGA CATCGGTTGC

V S D P V I I H F E V S M S Q V TGTCTGACCC AGTCATCATC CATTTCGAGG TGTCCATGTC TCAGCTGGTG ACAGACTGGG TCAGTAGTAG GTAAAGCTCC ACAGGTACAG AGTCGACCAC

K V D E V N Q 1 M B T H L L K Q AAGGTGGATG AAGTAAACCA GATCATGGAG ACCAACCTGT GGCTCAAGCA TTCCACCTAC TTCATTTGGT CTAGTACCTC TGGTTGGACA CCGAGTTCGT

I W N D Y K L K W N P S D Y G G AATCTGGAAT GACTACAAGC TGAAGTGGAA CCCCTCTGAC TATGGTGGGG TTAGACCTTA CTGATGTTCG ACTTCACCTT GGGGAGACTG ATACCACCCC

A E F R V P A Q K I W K P D I V CAGAGTTCAT GCGTGTCCCT GCACAGAAGA TCTGGAAGCC AGACATTGTG GTCTCAAGTA CGCACAGGGA CGTGTCTTCT AGACCTTCGG TCTGTAACAC

L Y N N A V G D F Q V D D K T K A CTGTATAACA ATGCTGTTGG GGATTTCCAG GTGGACGACA AGACCAAAGC GACATATTGT TACGACAACC CCTAAAGGTC CACCTGCTGT TCTGGTTTCG

L L K Y T G E V T I P P A I F CTTACTCAAG TACACTGGGG AGGTGACTTG GATACCTCCG GCCATCTTTA GAATGAGTTC ATGTGACCCC TCCACTGAAC CTATGGAGGC CGGTAGAAAT

K S S C K I D V T Y F P F D Y Q N AGAGCTCCTG TAAAATCGAC GTGACCTACT TCCCGTTTGA TTACCAAAAC TCTCGAGGAC ATTTTAGCTG CACTGGATGA AGGGCAAACT AATGGTTTTG

C T M K F G S S Y D K A K I D 1 TGTACCATGA AGTTCGGTTC CTGGTCCTAC GATAAGGCGA AAATCGATCT ACATGGTACT TCAAGCCAAG GACCAGGATG CTATTCCGCT TTTAGCTAGA

V I G S S M N L K D Y W E S G GGTCCTGATC GGCTCTTCCA TGAACCTCAA GGACTATTGG GAGAGCGGCG CCAGGACTAG CCGAGAAGGT ACTTGGAGTT CCTGATAACC CTCTCGCCGC

E A I I K A P G Y K H D I K Y N AGTGGGCCAT CATCAAAGCC CCAGGCTATA AACACGACAT CAAGTACAAC TCACCCGGTA GTAGTTTCGG GGTCCGATAT TTGTGCTGTA GTTCATGTTG C C E E I Y P D I T Y S L Y I R R TGCTGCGAGG AGATCTACCC CGACATCACA TACTCGCTGT ACATCCGGCG ACGACGCTCC TCTAGATGGG GCTGTAGTGT ATGAGCGACA TGTAGGCCGC P L F Y T I N L I I P C L I CCTGCCCTTG TTCTACACCA TCAACCTCAT CATCCCCTGC CTGCTCATCT GGACGGGAAC AAGATGTGGT AGTTGGAGTA GTAGGGGACG GACGAGTAGA

S F L T V L V F Y S D C G E K CCTTCCTCAC TGTGCTCGTC TTCTACCTGC CCTCCGACTG CGGTGAGAAG GGAAGGAGTG ACACGAGCAG AAGATGGACG GGAGGCTGAC GCCACTCTTC

V T C I S V L S L T V F V GTGACCCTGT GCATTTCTGT CCTCCTCTCC CTGACGGTGT TTCTCCTGGT CACTGGGACA CGTAAAGACA GGAGGAGAGG GACTGCCACA AAGAGGACCA

I T E T I P S T S L V I P I G GATCACTGAG ACCATCCCTT CCACCTCGCT GGTCATCCCC CTGATTGGAG CTAGTGACTC TGGTAGGGAA GGTGGAGCGA CCAGTAGGGG GACTAACCTC

E Y F T M I F V T L S I V I T AGTACCTCCT GTTCACCATG ATTTTTGTAA CCTTGTCCAT CGTCATCACC TCATGGAGGA CAAGTGGTAC TAAAAACATT GGAACAGGTA GCAGTAGTGG

V F V L N V H Y R T P T T H T M P GTCTTCGTGC TCAACGTGCA CTACAGAACC CCGACGACAC ACACAATGCC CAGAAGCACG AGTTGCACGT GATGTCTTGG GGCTGCTGTG TGTGTTACGG

S W V K T V F L K 1 P R V M F CTCATGGGTG AAGACTGTAT TCTTGAACCT GCTCCCCAGG GTCATGTTCA GAGTACCCAC TTCTGACATA AGAACTTGGA CGAGGGGTCC CAGTACAAGT

M T R P T S K E G N A Q K P R P TGACCAGGCC AACAAGCAAC GAGGGCAACG CTCAGAAGCC GAGGCCCCTC ACTGGTCCGG TTGTTCGTTG CTCCCGTTGC GAGTCTTCGG CTCCGGGGAG

Y G A E L S N L N C F S R A E S K TACGGTGCCG AGCTCTCAAA TCTGAATTGC TTCAGCCGCG CAGAGTCCAA ATGCCACGGC TCGAGAGTTT AGACTTAACG AAGTCGGCGC GTCTCAGGTT

G C K E G Y P C Q D G M C G Y C AGGCTGCAAG GAGGGCTACC CCTGCCAGGA CGGGATGTGT GGTTACTGCC TCCGACGTTC CTCCCGATGG GGACGGTCCT GCCCTACACA CCAATGACGG

H H I K I S F S A N L T R S ACCACCGCAG GATAAAAATC TCCAATTTCA GTGCTAACCT CACGAGAAGC TGGTGGCGTC CTATTTTTAG AGGTTAAAGT CACGATTGGA GTGCTCTTCG

S S S E S V D A V L S L S A L S P TCTAGTTCTG AATCTGTTGA TGCTGTGCTG TCCCTCTCTG CTTTGTCACC AGATCAAGAC TTAGACAACT ACGACACGAC AGGGAGAGAC GAAACAGTGG

E I K E A I Q S V K Y A E N M AGAAATCAAA GAAGCCATCC AAAGTGTCAA GTATATTGCT GAAAATATGA TCTTTAGTTT CTTCGGTAGG TTTCACAGTT CATATAACGA CTTTTATACT

K A Q N E A K E I Q D D W K Y V A AAGCACAAAA TGAAGCCAAA GAGATTCAAG ATGATTGGAA GTATGTTGCC TTCGTGTTTT ACTTCGGTTT CTCTAAGTTC TACTAACCTT CATACAACGG

M V I D R I F L V F T L V C I L ATGGTGATTG ATCGTATTTT TCTGTGGGTT TTCACCCTGG TGTGCATTCT TACCACTAAC TAGCATAAAA AGACACCCAA AAGTGGGACC ACACGTAAGA G T A G I F. L Q P L M A R E D A AGGGACAGCA GGATTGTTTC TGCAACCCCT GATGGCCAGG GAAGATGCAT TCCCTGTCGT CCTAACAAAG ACGTTGGGGA CTACCGGTCC CTTCTACGTA

Sequence Number 2 β4 subunit

M

ATGA ■TACT

R R A P S 1 V F F L V A L C G R GGCGCGCGCC TTCCCTGGTC CTTTTCTTCC TGGTCGCCCT TTGCGGGCGC CCGCGCGCGG AAGGGACCAG GAAAAGAAGG ACCAGCGG€A AACGCCCGCG

G N C R V A N A E E K 1 M D D L L GGGAACTGCC GCGTGGCCAA TGCGGAGGAA AAGCTGATGG ACGACCTTCT CCCTTGACGG CGCACCGGTT ACGCCTCCTT TTCGACTACC TGCTGGAAGA

N K T R Y H N L I R P A T S S S GAACAAAACC CGTTACAATA ACCTGATCCG CCCAGCCACC AGCTCCTCAC CTTGTTTTGG GCAATGTTAT TGGACTAGGC GGGTCGGTGG TCGAGGAGTG

Q L I S I K L Q L S L A Q L I S V AGCTCATCTC CATCAAGCTG CAGCTCTCCC TGGCCCAGCT TATCAGCGTG TCGAGTAGAG GTAGTTCGAC GTCGAGAGGG ACCGGGTCGA ATAGTCGCAC

N E R E Q I M T T N V W L K Q E AATGAGCGAG AGCAGATCAT GACCACCAAT GTCTGGCTGA AACAGGAATG TTACTCGCTC TCGTCTAGTA CTGGTGGTTA CAGACCGACT TTGTCCTTAC

T D Y R L T W N S S R Y E G V N GACTGATTAC CGCCTGACCT GGAACAGCTC CCGCTACGAG GGTGTGAACA CTGACTAATG GCGGACTGGA CCTTGTCGAG GGCGATGCTC CCACACTTGT

I L R I P A K R I W L P D I V L Y TCCTGAGGAT CCCTGCAAAG CGCATCTGGT TGCCTGACAT CGTGCTTTAC AGGACTCCTA GGGACGTTTC GCGTAGACCA ACGGACTGTA GCACGAAATG

N N A D G T Y E V S V Y T N L I V AACAACGCCG ACGGGACCTA TGAGGTGTCT GTCTACACCA ACTTGATAGT TTGTTGCGGC TGCCCTGGAT ACTCCACAGA CAGATGTGGT TGAACTATCA

R S N G S V L L P P A I Y K S CCGGTCCAAC GGCAGCGTCC TGTGGCTGCC CCCTGCCATC TACAAGAGCG GGCCAGGTTG CCGTCGCAGG ACACCGACGG GGGACGGTAG ATGTTCTCGC

A C K I E V K Y F P F D Q Q N C T CCTGCAAGAT TGAGGTGAAG TACTTTCCCT TCGACCAGCA GAACTGCACC GGACGTTCTA ACTCCACTTC ATGAAAGGGA AGCTGGTCGT CTTGACGTGG

L K F R S W T Y D H T E I D M V L CTCAAGTTCC GCTCCTGGAC CTATGACCAC ACGGAGATAG ACATGGTCCT GAGTTCAAGG CGAGGACCTG GATACTGGTG TGCCTCTATC TGTACCAGGA

M T P T A S M D D F T P S G E W CATGACGCCC ACAGCCAGCA TGGATGACTT TACTCCCAGT GGTGAGTGGG GTACTGCGGG TGTCGGTCGT ACCTACTGAA ATGAGGGTCA CCACTCACCC

D I V A L P G R R T V N P Q D P S ACATA6TGGC CCTCCCAGGG AGAAGGACAG TGAACCCACA AGACCCCAGC TGTATCACCG GGAGGGTCCC TCTTCCTGTC ACTTGGGTGT TCTGGGGTCG Y V D V T Y D F I I K R K P L F Y TACGTGGACG TGACTTACGA CTTCATCATC AAGCGCAAGC CTCTGTTCTA ATGCACCTGC ACTGAATGCT GAAGTAGTAG TTCGCGTTCG GAGACAAGAT

T I N L I P C V L T T L L A I CACCATCAAC CTCATCATCC CCTGCGTGCT CACCACCTTG CTGGCCATCC GTGGTAGTTG GAGTAGTAGG GGACGCACGA GTGGTGGAAC GACCGGTAGG

L V F Y L P S D C G E K M T L C I TCGTCTTCTA CCTGCCATCC GACTGCGGCG AGAAGATGAC ACTGTGCATC AGCAGAAGAT GGACGGTAGG CTGACGCCGC TCTTCTACTG TGACACGTAG

S V L L A L T F F 1 L L I S K I V TCAGTGCTGC TGGCACTGAC ATTCTTCCTG CTGCTCATCT CCAAGATCGT AGTCACGACG ACCGTGACTG TAAGAAGGAC GACGAGTAGA GGTTCTAGCA

P P T S L D V P L I G K Y L M F GCCACCCACC TCCCTCGATG TGCCTCTCAT CGGCAAGTAC CTCATGTTCA CGGTGGGTGG AGGGAGCTAC ACGGAGAGTA GCCGTTCATG GAGTACAAGT

T M V L V T F S I V T S V C V L N CCATGGTGCT GGTCACCTTC TCCATCGTCA CCAGCGTCTG TGTGCTCAAT GGTACCACGA CCAGTGGAAG AGGTAGCAGT GGTCGCAGAC ACACGAGTTA

V H H R S P S T H T "M A P V K R GTGCACCACC GCTCGCCCAG CACCCACACC ATGGCACCCT GGGTCAAGCG CACGTGGTGG CGAGCGGGTC GTGGGTGTGG TACCGTGGGA CCCAGTTCGC

C F L H K L P T F L F M K R P G CTGCTTCCTG CACAAGCTGC CTACCTTCCT CTTCATGAAG CGCCCTGGCC GACGAAGGAC GTGTTCGACG GATGGAAGGA GAAGTACTTC GCGGGACCGG

P D S S P A R A F P P S K S C V T CCGACAGCAG CCCGGCCAGA GCCTTCCCGC CCAGCAAGTC ATGCGTGACC GGCTGTCGTC GGGCCGGTCT . CGGAAGGGCG GGTCGTTCAG TACGCACTGG

K P E A T A T S T S P S N F Y G N AAGCCCGAGG CCACCGCCAC CTCCACCAGC CCCTCCAACT TCTATGGGAA TTCGGGCTCC GGTGGCGGTG GAGGTGGTCG GGGAGGTTGA AGATACCCTT

S M Y F V N P A S A A S K S P A CTCCATGTAC TTTGTGAACC CCGCCTCTGC AGCTTCCAAG TCTCCAGCCG GAGGTACATG AAACACTTGG GGCGGAGACG TCGAAGGTTC AGAGGTCGGC

G S T P V A I P R D F L R S S G GCTCTACCCC GGTGGCTATC CCCAGGGATT TCTGGCTGCG GTCCTCTGGG CGAGATGGGG CCACCGATAG GGGTCCCTAA AGACCGACGC CAGGAGACCC

R F R Q D V Q E A L E G V S F I A AGGTTCCGAC AGGATGTGCA GGAGGCATTA GAAGGTGTCA GCTTCATCGC TCCAAGGCTG TCCTACACGT CCTCCGTAAT CTTCCACAGT CGAAGTAGCG

Q H M K N D D E D Q S V V E D CCAGCACATG AAGAATGACG ATGAAGACCA GAGTGTCGTT GAGGACTGGA GGTCGTGTAC TTCTTACTGC TACTTCTGGT CTCACAGCAA CTCCTGACCT

K Y V A M V V D R L F L V F M F AGTACGTGGC TATGGTGGTG GACCGGCTGT TCCTGTGGGT GTTCATGTTT TCATGCACCG ATACCACCAC CTGGCCGACA AGGACACCCA CAAGTACAAA

V C V L G T V G L F L P P L F Q T GTGTGCGTCC TGGGCACTGT GGGGCTCTTC CTACCGCCCC TCTTCCAGAC CACACGCAGG ACCCGTGACA CCCCGAGAAG GATGGCGGGG AGAAGGTCTG H A A E G P Y A A Q R D

CCATGCAGCT TCTGAGGGGC CCTACGCTGC CCAGCGTGAC TGA GGTACGTCGA AGACTCCCCG GGATGCGACG GGTCGCACTG ACT

Claims

1. A polypeptide chain which comprises four subunits of a ligand-gated ion channel and is able to form a functional ligand-gated ion channel in combination with another subunit.

2. A polypeptide chain which comprises five subunits of a ligand-gated ion channel and is able to form a functional ligand-gated ion channel.

3. A polypeptide according to claim 1 or claim 2 wherein at least one of the subunits binds to one of acetylcholine (ACH), γ-aminobutyric acid (GABA) , glycine and serotonin (5HT).

4. A polypeptide according to any of claims 1 to 3, comprising α and β subunits.

5. A polypeptide according to claim 4, comprising two α and three β subunits.

6. A polypeptide according to claim 5, comprising two identical α subunits and three identical β subunits.

7. A polypeptide according to claim 6, comprising α3 and β4 subunits.

8. A polypeptide according to claim 7, wherein the subunits are connected in the order β4-β4-α3-β4-α3.

9. A polypeptide according to any one of the preceeding claims, wherein the subunits are connected via linker regions.

10. A polynucleotide encoding a polypeptide according to any one of claims 1 to 9.

11. A polynucleotide according to claim 10, wherein the polynucleotide is a mRNA molecule.

12. A polynucleotide according to claim 11 , wherein the polynucleotide is a capped mRNA molecule.

13. A cell transformed with a capped mRNA molecule according to claim 12.

14. A vector comprising a polynucleotide according to claim 10.

15. A cell transformed with a vector according to claim 14.

16. A method for obtaining a homogenous population of ligand-gated ion channels comprising expressing a polynucleotide according to claim 10.

17. A method according to claim 16, wherein the polynucleotide is expressed in a cell which does not normally produce ligand-gated ion channels.

18. A method according to claim 17, wherein the polynucleotide is expressed in an oocyte.

19. A method according to claim 18, wherein the polynucleotide is expressed in a Xenopus oocyte.

20. A method for assaying actives comprising; a) producing a homogeneous population of ligand-gated ion channels produced from a polypeptide according to, any one of claims 1 to 9, b) bringing the ligand-gated ion channels into contact with a ligand to generate a response, c) bringing the ligand-gated ion channels into contact with an active, d) detecting and measuring any electrical response which is generated across the ligand-gated ion channel.

21. A method according to claim 20, wherein the ligand of step b) and the active of step c) are the same substance.

22. An assay according to claim 20 or claim 21 , wherein step a) comprises expressing a polynucleotide according to claim 10.

23. A method for introducing a mutation into one copy of a subunit appearing in a ligand-gated ion channel in a plurality of copies comprising introducing the mutation into a polynucleotide according to claim 10.