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WO2004056867A2 - Nouvel outil pour tester des substances pharmacologiques actives - Google Patents

Nouvel outil pour tester des substances pharmacologiques actives Download PDF

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Publication number
WO2004056867A2
WO2004056867A2 PCT/GB2003/005502 GB0305502W WO2004056867A2 WO 2004056867 A2 WO2004056867 A2 WO 2004056867A2 GB 0305502 W GB0305502 W GB 0305502W WO 2004056867 A2 WO2004056867 A2 WO 2004056867A2
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ligand
subunits
gated ion
polynucleotide
ion channel
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WO2004056867A3 (fr
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Lucia Sivilotti
Paul Groot-Kormelink
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University College London
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University College London
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • 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 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.
  • the neuronal nicotinic receptors are generally composed of two ⁇ and three ⁇ subunits. So far nine different ⁇ subunits have been identified ( ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ , ⁇ 9 and ⁇ 10) and three ⁇ subunits ( ⁇ 2, ⁇ 3 and ⁇ 4).
  • GABA receptors are believed to be composed of subunits from six classes each with several isoforms ( ⁇ 1-6, ⁇ 1-3, Y 1-3, ⁇ , ⁇ , ⁇ , ⁇ ). ( Le Novere et al, Inc.
  • Neuronal nicotinic receptors can be produced by expressing one subunit only (if the subunit is ⁇ 7 or ⁇ 9), by expressing an ⁇ and a ⁇ subunit together ( ⁇ 2- ⁇ 4 plus ⁇ 2 or ⁇ 4) or in a triplet fashion by expressing an ⁇ together with ⁇ and either ⁇ 5 or ⁇ 3.
  • dimer constructs were created by linking polynucleotides encoding ⁇ 3 and ⁇ 4 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 ⁇ 3- ⁇ 4 orientation failed to produce functional receptors either when expressed alone or along with any monomer.
  • Dimers of the ⁇ 4- ⁇ 3 orientation did not produce functional receptors when expressed alone or along with ⁇ 3, ⁇ 4, ⁇ 6 or ⁇ 3 subunits (small responses were observed with monomer ⁇ 2, ⁇ 5 or ⁇ 2 subunits).
  • ⁇ 4- ⁇ 3 tandem was expressed along with ⁇ 4 subunits, large inward currents were observed in response to acetylcholine.
  • the receptor expressed from dimers with monomer ( ⁇ + ⁇ ) did not have the composition expected.
  • the expected composition is two ⁇ subunits from the dimers and three ⁇ subunits, two from the dimers and one from a monomer.
  • 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 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".
  • 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).
  • 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.
  • the preferred embodiment of this invention is focussed on nicotinic acetylcholine receptors formed from ⁇ and ⁇ subunits.
  • nicotinicoid receptors nicotinic receptors, GABA A and GABA C receptors, glycine receptors, 5-HT 3 receptors and some glutamate activated anionic channels
  • LGICdb provides an interface to programs capable of screening sequences and indicating similarities between them).
  • Subunits designated as ⁇ 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 ⁇ 1 subunit. Subunits lacking the pair of adjacent cysteine residues are non- ⁇ subunits, for example ⁇ subunits.
  • Sequence number 1 , ⁇ 3 subunit and sequence ID Nos. 1 and 2 provide the DNA and protein sequence of an ⁇ 3 subunit.
  • ⁇ 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, ⁇ 4 subunit and sequence ID Nos. 3 and 4 provide the DNA and protein sequence of a ⁇ 4 subunit.
  • ⁇ 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.
  • similarity refers to a sequence comparison based on identical matches between correspondingly indentical positions in the sequences being compared.
  • 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.
  • 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.
  • a channel is formed from two identical ⁇ subunits and three identical ⁇ subunits. More preferably the channel is formed from ⁇ 3 and ⁇ 4 subunits.
  • the channel is formed from subunits which are connected in the order ⁇ 4- ⁇ 4- ⁇ 3- ⁇ 4- ⁇ 3.
  • linker regions can vary widely in their length and composition.
  • 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.
  • 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.
  • a vector comprising the polynucleotide would be a form suitable for transfections of other cells.
  • 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.
  • the polynucleotide would be expressed in a heterologous system.
  • 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.
  • 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.
  • the ligand to which the ligand-gated ion channel responds and the active are the same substance.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 3- ⁇ 4 dimer construct
  • Figure 4 shows creation of ⁇ 4- ⁇ 3 dimer construct
  • Figure 5 shows subcloning of ⁇ 3- ⁇ 4 dimer construct into an alternative plasmid
  • Figure 6 shows subcloning of ⁇ 4- ⁇ 3 dimer construct into an alternative plasmid
  • Figure 7 shows further subcloning of ⁇ 3- ⁇ 4 dimer construct into another plasmid
  • Figure 8 shows further subcloning of ⁇ 4- ⁇ 3 dimer construct into a different plasmid
  • Figure 9 shows creation of ⁇ 4- ⁇ 4- ⁇ 3 trimer construct
  • Figure 10 shows creation of ⁇ 4- ⁇ 4- ⁇ 3- ⁇ 4- ⁇ 3 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.
  • This example describes a method to obtain a ligand-gated ion channel according to the present invention.
  • cDNAs for the human neuronal nicotinic acetylcholine receptor subunits ⁇ 3 and ⁇ 4 (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 ⁇ -globin regions (Akopian et al., 1996) for oocyte expression studies) and pcDNA3.1 + (mammalian expression vector, Invitrogen, The Netherlands).
  • plasmid constructs will be referred to as ⁇ 3/pSP64GL [1], ⁇ 4/pSP64GL [2], ⁇ 3/pcDNA3.1 [3], and ⁇ 4/pcDNA3.1 [4] respectively.
  • Both cDNAs were also cloned into the epitope-tagged vector pcDNA3.1_myc-His version C (Invitrogen, The Netherlands).
  • the stop codons of the ⁇ 3 and ⁇ 4 subunit cDNAs have to be removed.
  • the ⁇ 3 and ⁇ 4 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 ⁇ 3 and ⁇ 4 are [EcoRI - GCCACCATG....CDS ( ⁇ 3 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 ⁇ 3/pcDNA3.1_myc-His [5] and ⁇ 4/pcDNA3.1_myc-His [6], respectively.
  • Agel site The Agel site is uniquely situated on the pcDNA3.1/t ⁇ 7yc-His version C vector between the epitope tag sequences of myc and His.
  • the dimer construct is created by a ligation reaction of three DNA fragments (see Figures 3 and 4).
  • the resulting dimer constructs are named ⁇ 3_ ⁇ 4/pcDNA3.1_myc-His [7] and ⁇ 4_ ⁇ 3/pcDNA3.1_ yc-His [8].
  • the dimer constructs are named ⁇ 3_ ⁇ 4/pcDNA3.1 [9] and ⁇ 4_ ⁇ 3/pcDNA3.1 [10].
  • T7 promotor to the BGH pA sequence were found to be 100% correct.
  • trimer was constructed by ligation of three DNA fragments originating from the following digests; ⁇ 4_ ⁇ 3/pSP64GL [12, digested with Xbal and
  • 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.
  • neutralised ethyl m-aminobenzoate solution tricaine, methanesulphonate salt; 0.2% solution weight/volume; Sigma Chemical Co.
  • Clumps of stage V-VI oocytes were dissected in a sterile modified Barth's solution of composition (in mM): NaCI 88; KC1 1 ; MgCI 2 0.82; CaCI 2 0.77; NaHC0 3 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 ⁇ 3 and ⁇ 4s.
  • 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, MgCI 2 2 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 M ⁇ on the current-passing side.
  • 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 EC 20 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
  • n H is the Hill coefficient.
  • CVFIT 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 , EC 50 and n H . The means and standard deviation of the means for each combination are shown in Table 1. Tablel ; EC 50 values were obtained by fitting separately the individual concentration-response curves.
  • 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

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Abstract

La présente invention concerne des canaux ioniques à portes-ligands appartenant à la superfamille des nicotiniques, et des procédés pour limiter leur composition en sous-unités. L'invention a pour objet une chaîne polypeptidique comprenant quatre ou cinq des sous-unités d'un canal ionique à portes-ligands. Cette chaîne polypeptidique est capable de former un canal ionique à portes-ligands fonctionnel en combinaison avec une autre sous-unité ou de manière autonome respectivement.
PCT/GB2003/005502 2002-12-19 2003-12-18 Nouvel outil pour tester des substances pharmacologiques actives Ceased WO2004056867A2 (fr)

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AU2003295126A AU2003295126A1 (en) 2002-12-19 2003-12-18 Ion channel for testing pharmacological active compounds

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GB0229586A GB0229586D0 (en) 2002-12-19 2002-12-19 Novel tool for testing pharmacological actives
GB0229586.3 2002-12-19

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111556751A (zh) * 2017-11-10 2020-08-18 霍华休斯医学研究院 经修饰的配体门控的离子通道及使用方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BAUMANN SABINE W ET AL: "Forced subunit assembly in alpha1beta2gamma2 GABAA receptors. Insight into the absolute arrangement." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 48, 29 November 2002 (2002-11-29), pages 46020-46025, XP002279648 ISSN: 0021-9258 cited in the application *
BAUMANN SABINE W ET AL: "Subunit arrangement of gamma-aminobutyric acid type A receptors" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 39, 28 September 2001 (2001-09-28), pages 36275-36280, XP002279649 ISSN: 0021-9258 cited in the application *
BROADBENT S ET AL.: "Human recombinant neuronal nicotinic receptors: expression of tandem subunit constructs" JOURNAL OF PHYSIOLOGY, vol. 547P, 2003, page 13P, XP002279647 *
GROOT-KORMELINK PAUL J ET AL: "Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: A reporter mutation approach" BRITISH JOURNAL OF PHARMACOLOGY, vol. 134, no. 4, October 2001 (2001-10), pages 789-796, XP002279650 ISSN: 0007-1188 *
PRYBYLOWSKI K. ET AL.: "Tandem Proteins to study NMDA receptor stoichiometry" SOCIETY FOR NEUROSCIENCE, vol. 25, 1999, page 1487, XP002279651 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111556751A (zh) * 2017-11-10 2020-08-18 霍华休斯医学研究院 经修饰的配体门控的离子通道及使用方法
EP3706743A4 (fr) * 2017-11-10 2022-05-11 Howard Hughes Medical Institute Canaux ioniques modifiés sensibles à un ligand et méthodes d'utilisation
US12247064B2 (en) 2017-11-10 2025-03-11 Howard Hughes Medical Institute Modified ligand-gated ion channels and methods of use
AU2018364738B2 (en) * 2017-11-10 2025-03-13 Howard Hughes Medical Institute Modified ligand-gated ion channels and methods of use

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AU2003295126A8 (en) 2004-07-14
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