JP2003512830A - DNA molecule encoding L-glutamate-dependent chloride channel from cystcelka americana - Google Patents

Abstract

  (57) [Summary] The present invention relates, in part, to an isolated nucleic acid molecule (polynucleotide) encoding a Sistocerca americana (a herbivorous orthoptera) glutamate-gated chloride channel. The present invention also provides a recombinant vector and a recombinant host containing a DNA fragment encoding a cystcelca americana glutamate-dependent chloride channel, a substantially purified form of a related cystcelka americana glutamate-dependent chloride channel, and the like. And related methods related to the identification of compounds that modulate related cystselca americana glutamate-gated chloride channels, which may be useful as insecticides.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention is, in part, in Schistocerciana americana (Schistocerc).
a americana, grasshopper) (grasshopper)
per, herbivorous insects)) and an isolated nucleic acid molecule (polynucleotide) encoding a glutamate-gated chloride channel. The present invention also relates to recombinant vectors and recombinant hosts containing a DNA fragment encoding a S. americana glutamate-gated chloride channel, a substantially purified form of the relevant Cystoluca americana (S. (America) glutamic acid-gated chloride channels and recombinant membrane fractions containing these proteins, related muteins, and compounds that regulate the related S. americana glutamic acid-gated chloride channels, which are insecticides. Will be useful as).

BACKGROUND OF THE INVENTION Dudel et al. (1989, Brain Res. 481: 215-220) described the arthropod Schistocerca gregaria.
Direct gating of ion channels by glutamate in the leg muscle of ria) (locust).

Glutamate-gated chloride channels (or H receptors) have been identified in arthropod nerves and muscles (Lingle et al., 1981, Brai).
n Res. 212: 481-488; Horseman et al., 1988, Neu.
rosci. Lett. 85: 65-70; Wafford and Sattel.
le, 1989, J.I. Exp. Bio. 144: 449-462; Lea and Userwood, 1973, Comp. Gen. Palmacol. 4: 3
33-350; and Cull-Candy, 1976, J. Am. Physiol.
255: 449-464).

[0004] The glutamate-gated chloride ion channel is also found in the soil nematode Caenorhabditis elegans (Cul).
ly et al., 1994, Nature 371: 707-711; US Pat. No. 5,5.
27,703 and Arena et al., 1992, Molecular Brai.
n Research. 15: 339-348) and Drosophila melanogaster (Cul.
ly et al., 1996, J. Am. Biol. Chem. 271: 20187-20191
) Has been cloned from.

Raymond et al. (2000, Neuroreport 11: 2695-2.
701) is Periplanta americana
Cana) (cockroaches) discloses the detection of GluCl channels in dorsal midline neurons.

The invertebrate glutamate-gated chloride channels are important targets for the widely used class of avermectin, an anthelmintic and insecticidal compound. Avermectin was initially the actinomycete Streptomyces avermitilis (Strepto
myces avermitilis) is a family of macrocyclic lactones. Ivermectin, a semi-synthetic avermectin derivative (22,23-
Dihydro-avermectin B _1a ) is used worldwide for treating human and animal parasitic helminths and pests. Avermectin is still
The most potent broad-spectrum endocide (en) with low toxicity to the host
It is. Even after being used for many years in this field,
Little resistance to avermectin has occurred in insect populations. The combination of good therapeutic index and low resistance strongly suggests that glutamate-gated chloride (GluCl) channels are still good targets for insecticide development.

Despite the identification of said cDNA clones encoding GluCl channels, insecticidal, acaricidal and / or acaricidal for animal health or crop protection.
Alternatively, it would be advantageous to identify additional invertebrate genes encoding GluCl channels to allow screening to identify novel GluCl channel modulators that may have nematicidal activity. The present invention is a cystocerca americana Glu.
An isolated nucleic acid molecule expressing a Gl channel (expression of grasshopper GluCl cRNA in Xenopus oocytes or other suitable host cells is not
The disclosure of (providing uCl channels) addresses these requirements and fulfills them. Cystselka Americana (Schis
Heterologous expression of the Tocca americana (grasshopper) GluCl channel will allow pharmacological analysis of compounds active against parasitic invertebrate species associated with animal and human health. Such species include helminths,
Includes fleas, ticks and lice. Heterologous cell lines expressing active GluCl channels can be used to establish functional or binding assays to identify novel GluCl channel modulators that may be useful in the control of said species.

SUMMARY OF THE INVENTION The present invention provides a novel Schistocerca americana.
(Grasshopper) isolated or purified nucleic acid molecule (polynucleotide) encoding an invertebrate GluCl channel protein.

The present invention provides a novel Schistocerca americana (Schistocerca a)
(Grasshopper) isolated or purified nucleic acid molecule (polynucleotide) encoding an mRNA expressing an invertebrate GluCl channel protein. This DNA molecule is referred to herein as SEQ ID NO: (SEQ ID
NO) 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9.

The invention also provides a novel Schistocerca americana.
(Grasshopper) biologically active fragments or mutants of SEQ ID NOs: 1, 3, 5, 7 and 9 which encode an mRNA expressing an invertebrate GluCl channel protein. Any such biologically active fragments and / or mutants are grasshopper GluCl channel proteins (grasshoppers set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10). Will encode either proteins or protein fragments that substantially mimic the pharmacological properties of GluCl channel proteins (including but not limited to GluCl channel proteins). Any such polynucleotides include, but are not necessarily limited to, nucleotide substitutions, deletions, additions, amino-terminal truncation errors, and carboxy-terminal truncation errors (these mutations are referred to as Xenopus. ) Functional in a eukaryotic cell such as an oocyte Schistocerca americana
ana) encodes an mRNA expressing the GluCl channel and encodes Sistocerca americana Glu
It may be useful for screening for agonists and / or antagonists of Cl activity).

A preferred aspect of this aspect of the invention is the novel Schistocerca americana (disclosed in Figures 1A-F).
Grasshopper) cDNA molecule encoding GluCl protein (SEQ ID NO: 1)
3, 5, 5 and 9).

Isolated nucleic acid molecules of the present invention include deoxyribonucleic acid molecules (DNA), such as genomic DNA and complementary DNA (cDNA) [which may be single-stranded (coding or non-coding) or double-stranded. , As well as synthetic DNA, such as synthetic single-stranded polynucleotides. In addition, the isolated nucleic acid molecule of the present invention may include a ribonucleic acid molecule (RNA).

The present invention also relates to recombinant vectors and recombinant hosts (both prokaryotic and eukaryotic) containing the substantially purified nucleic acid molecules disclosed throughout this specification.

The invention also provides recombinant host cells (both prokaryotic and eukaryotic, as well as stable and transient traits) containing functional and processed proteins encoded by the nucleic acids of the invention. Transformed cells) subcellular membrane fraction. The present invention is a cystocerca americana.
a americana) contaminating proteins comprising GluCl channels (
Other cystocerca americana
na) or a substantially purified membrane preparation substantially free of proteins (including but not limited to host proteins from recombinant cells expressing SaGluCl or SaGluCl2). Particularly preferred is the full length G disclosed in Figures 2A-B and set forth in SEQ ID NOs: 2, 4, 6, 8 and 10.
Schistocerca americana containing GluCl protein containing functional form of luCl channel protein
) Membrane preparation containing GluCl channels. To this end, the invention also provides a functional and fully processed Cystoluca americana (
Substantially purified membrane purified from a recombinant host (regardless of whether it is a recombinant eukaryotic host or a recombinant prokaryotic host) in which the recombinant vector expresses the Schistocerca americana) GluCl channel Regarding the preparation. Particularly preferred are SEQ ID NOs: 2, 4, 6, 8 and 10 disclosed in Figures 2A-B.
The functional sequence of the full-length SaGluCl protein described in S. et al. Is included in the protein sequence Schistocerca americana
a) Membrane preparation containing recombinant forms of GluCl channels, SaGluCl1, SaGluCl2 or any heteromultimer combination. These subcellular membrane fractions were isolated from Schistocerca americana.
na) contains wild-type or mutant forms that are biologically functional forms of GluCl channels, SaGluCl1, SaGluCl2 or any heteromultimeric combination thereof at levels substantially above endogenous levels, and It may be useful in the various assays described throughout the specification. A preferred eukaryotic host suitable for expressing the glutamate-gated channels of the present invention is the Xenopus oocyte.

Xenopus oocytes or Chinese hamster ovaries (
It is exemplified herein that expression of any DNA molecule encoding the short form of SaGluCl1 (ie, SEQ ID NO: 5 and / or 7) in CHO) cells confers a functional GluCl channel. In contrast, SaGl
Co-expression of a short form of uCl1 with SaGluCl2 (eg from SEQ ID NO: 9) prevents the formation of a functional channel that is detectable when the short form of SaGluCl1 is expressed alone. Expression of either long form of SaGluCl1 has now been shown to be non-functional in Xenopus oocytes. The short form exemplified herein is the "SC" form (expression of a precursor protein containing the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, Xenopus () resulting in formation of the functional GluCl channel. Xeno
pus) expression in oocytes or CHO cells).

The present invention also includes S. americana, which comprises the amino acid sequences disclosed in Figures 2A-B and set forth in SEQ ID NOs: 2, 4, 6, 8 and 10.
a) relates to a substantially purified form of GluCl channel protein.

A preferred embodiment of this aspect of the invention is disclosed in FIGS.
Cystoluca americana consisting of the amino acid sequences described in
S. americana) GluCl channel protein.

Another preferred embodiment of the present invention is a recombinant containing a DNA expression vector comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7 and / or 9 and expressing the respective SaGluCl precursor protein. It relates to a substantially purified, fully processed GluCl channel protein obtained from a host cell. The recombinant host may be a mammalian cell line or Xenopu (Xenopu) as described above.
s) It is especially preferred that it is a eukaryotic host cell such as an oocyte.

Another preferred embodiment of the present invention comprises a complete open reading frame as set forth in SEQ ID NOs: 1, 3, 5, 7 and / or 9 which is appropriately expressed to functionalize each SaGluCl channel. To a substantially purified membrane preparation obtained from a recombinant host cell transformed or transfected with a DNA expression vector giving a processed form. It is particularly preferred that the recombinant host cell is a eukaryotic host cell such as a mammalian cell line or a Xenopus oocyte as described above.

The present invention also includes (but is not necessarily limited to) amino acid substitutions, deletions, additions, amino-terminal truncation errors and carboxy-terminal truncation errors.
These mutations confer a diagnostic, therapeutic or prophylactic protein or protein fragment, and are used in S. americana GluCl
Biological of S. americana GluCl channel proteins, which may be useful in screening for selective modulators, including but not limited to agonists and / or antagonists of channel pharmacology Active fragments or mutants (SEQ ID NO: 2, 4)
, 6, 8 and / or 10).

A preferred embodiment of the present invention is disclosed in Figures 2A-B and has SEQ ID NOs: 2, 4, 6
, 8 and 10 (respective amino acid sequences encoding grasshopper GluCl protein). One or more features of these channel proteins include screening methods for identifying novel GluCl channel modulators that may have insecticidal, acaricidal and / or nematicidal activity for animal health or crop protection. to enable. As mentioned above, Cystselka Americana (S
chistocerca americana) (grasshopper) GluCl
Heterologous expression of channels will allow pharmacological analysis of compounds against parasitic invertebrate species associated with animals and health. Such species include helminths, fleas, ticks and lice. Functional SaGluCl channels (eg,
A heterologous cell line expressing a functional form of SEQ ID NOS: 6 and 8, including (but not limited to) a mature form produced by heterologous expression of SEQ ID NO: 5 or 7, is used to It is possible to establish functional or binding assays to identify novel GluCl channel modulators that may be useful in control. Also, co-expression of a functional SaGluCl1 protein with SaGluCl2 confers a dominant inhibitory effect on channel activity, which is also useful in various assays utilized to identify modulators of in vivo GluCl channels.
Giving luCl2.

The present invention also relates to polyclonal and monoclonal antibodies raised against the form of SaGluCl or biologically active fragments thereof.

The present invention also provides fusion constructs expressing portions of SaGluCl and SaGlu2 linked to a variety of markers including, but not limited to, GFP (green fluorescent protein), MYC epitope and GST. Including (but not limited to) SaGluCl1 and / or SaGlu
2 fusion constructs. Any such fusion construct is expressed in the cell line of interest and can be used in a screen for one or more modulators of the SaGluCl protein disclosed herein.

The present invention provides methods of expressing the grasshopper GluCl channel protein bioisosteres disclosed herein, assays using these gene products, recombinant host cells containing DNA constructs expressing these proteins. , And GluCl
It relates to compounds identified by these assays that act as agonists or antagonists of channel activity.

One object of the present invention is to isolate a novel form of grasshopper GluCl or an isolated nucleic acid molecule (eg sequence of SEQ Nos. 1, 3, 5, 7 and 9). Any such polynucleotide has nucleotide substitutions, deletions, additions, amino-terminal truncation errors and carboxy-terminal truncation errors (these mutations are
MRNA expressing a protein or protein fragment for diagnosis, treatment or prevention
, Which may be useful in screening for selective modulators of invertebrate GluCl pharmacology), but is not necessarily limited thereto.

Another object of the invention is to provide a grasshopper GluCl protein or protein fragment encoded by the nucleic acid molecule described in the preceding paragraph.

Another object of the present invention is to provide a recombinant vector and a recombinant host cell containing a nucleic acid sequence encoding grasshopper GluCl protein or a bioisostere thereof.

One object of the present invention is to provide a substantially purified form of grasshopper GluCl protein set forth in SEQ ID NOs: 2, 4, 6, 8 and 10.

Another object of the invention is to include the complete open reading frame as set forth in SEQ ID NOs: 1, 3, 5, 7 and / or 9 to properly express it to functionalize the respective SaGluCl channels. It is to provide a substantially purified recombinant form of the grasshopper GluCl protein obtained from a recombinant host cell transformed or transfected with a DNA expression vector giving a processed form. It is particularly preferred that the recombinant host cell is a eukaryotic host cell line, such as a mammalian cell line.

One object of the present invention includes, but is not limited to, amino acid substitutions, deletions, additions, amino-terminal truncation errors and carboxy-terminal truncation errors.
These mutations provide a diagnostic or therapeutic and / or prophylactic protein or protein fragment), biologically of grasshopper GluCl protein as set forth in SEQ ID NOs: 2, 4, 6, 8 and 10. To provide active fragments and / or mutants.

Another object of the invention is a substantially purified subcellular membrane preparation comprising pharmacologically active grasshopper GluCl channels, in particular SEQ ID NOs: 2, 4, 6,
8 and 10 and to provide a subcellular fraction obtained from a host cell transfected or transformed with a DNA vector containing a nucleotide sequence encoding a protein comprising the amino acid set forth in Figures 2A-B.

Another object of the present invention is to include the complete open reading frame set forth in SEQ ID NOs: 1, 3, 5, 7 and / or 9 to express it appropriately to functionalize each SaGluCl channel. It is to provide a substantially purified membrane preparation obtained from a recombinant host cell transformed or transfected with a DNA expression vector giving a processed form. It is particularly preferred that the recombinant host cell is a eukaryotic host cell line such as a mammalian cell line or a Xenopus oocyte.

Another object of the present invention is grasshopper G containing grasshopper GluCl protein or bioisostere to screen for modulators of grasshopper GluCl channel activity, preferably selective modulators.
In using luCl protein or membrane preparations. Any such compound may be useful in the screening and selection of compounds active against parasitic invertebrate species of animal and health relevance. Such species include helminths, fleas, ticks and lice. These membrane preparations can be produced from heterologous cell lines expressing these GluCl and can constitute full-length proteins, biologically active fragments of said full-length proteins. Alternatively, it can be based on fusion proteins expressed from various fusion constructs that can be constructed with materials available in the art.

“Substantially free of other nucleic acids” as used in the present invention is at least 90%, preferably 95%, more preferably 99%, even more preferably 99% of other nucleic acids.
． It is 9% free (that is, such a proportion does not contain other nucleic acids). Therefore, grasshopper GluC substantially free of other nucleic acids
The l DNA preparation will contain no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1% non-grasshopper GluCl nucleic acid as a percentage of its total nucleic acid. Let's do it. Whether a given grasshopper GluCl DNA preparation is substantially free of other nucleic acids depends on nucleic acid purity, eg agarose gel electrophoresis, in combination with a suitable staining method (eg ethidium bromide staining). It can be determined by conventional techniques for evaluation or by sequencing.

As used herein, "substantially free of other proteins" or "substantially purified" means at least 90%, preferably 95%, more preferably 99%, even more from other proteins. It is preferably 99.9% free (ie such percentage does not contain other proteins). Therefore, the grasshopper GluCl protein preparation substantially free of other proteins is 10% or less, preferably 5% or less, more preferably 1% or less, and even more preferably 0.1% as a percentage of the total protein. % Or less non-grasshopper G
It will contain luCl protein. A given grasshopper Glu
Whether the Cl protein preparation is substantially free of other proteins is determined by eg sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with a suitable detection method (eg silver staining or immunoblotting). Can be determined by a conventional technique for evaluating protein purity such as. "Isolated grasshopper GluCl protein" or "purified grasshopper GluCl protein" used interchangeably with the terms "substantially free of other proteins" or "substantially purified" in the present invention. The term also means grasshopper GluCl protein isolated from natural sources. Use of the term "isolated" or "purified" indicates that grasshopper GluCl protein has been removed from its normal cellular environment. Thus, the isolated grasshopper GluCl protein may be present in a cell-free solution, or in a cellular environment different from that in which it naturally occurs. The term isolated does not mean that the isolated grasshopper GluCl protein is the only protein present, but rather the isolated grasshopper GluCl protein.
It means that the Cl protein is substantially free of other proteins and non-amino acid substances (eg, nucleic acids, lipids, carbohydrates) naturally associated with the grasshopper GluCl protein in vivo. For example, Grasshopper Glu
Cl proteins recombinantly expressed in prokaryotic or eukaryotic cells and substantially purified from this host cell that does not naturally express (ie, in the absence of intervention) this GluCl protein. Is, of course, an "isolated grasshopper GluCl protein", under any of the circumstances described herein.
As described above, the grasshopper GluCl protein preparation, which is an isolated or purified grasshopper GluCl protein, is substantially free of other proteins and is 10% or less, preferably 5% or less as a percentage of the total protein. Below, more preferably less than 1%, even more preferably less than 0.1% will contain non-grasshopper GluCl protein.

A “functional equivalent” or “biologically active equivalent” used interchangeably herein is a naturally occurring grasshopper GluCl by alternative splicing, deletion, mutation, substitution or addition. Means a protein that does not have the exact same amino acid sequence as, but retains substantially the same biological activity as grasshopper GluCl. Such functional equivalents would have significant amino acid sequence identity to the naturally occurring grasshopper GluCl. Genes and cDNAs encoding such functional equivalents are found in the naturally occurring grasshopper GluC.
It can be detected by low stringency hybridization to the DNA sequence encoding 1. For example, the naturally occurring grasshopper GluCl disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 2 and is encoded by SEQ ID NO: 1. A nucleic acid encoding a functional equivalent has at least about 50% identity at the nucleotide level to SEQ ID NO: 1.

As used in the present invention, “conservative amino acid substitution” means that one amino acid residue is replaced by another chemically similar amino acid residue. Specific examples of such conservative substitution include substitution between hydrophobic residues (isoleucine, leucine, valine or methionine), substitution of one polar residue with another polar residue of the same charge (eg lysine To arginine, and aspartic acid to glutamic acid).

“GluCl” used in the present invention means an L-glutamic acid-gated chloride ion channel.

The term “mammal” as used herein refers to any mammal, including humans.

BRIEF DESCRIPTION OF THE FIGURES In FIGS. 1A-F, the nucleotide sequences of DNA molecules encoding the respective grasshopper GluCl proteins set forth in SEQ ID NOs: 1, 3, 5, 7 and 9 are compared.

2A-B show grasshopper G as set forth in SEQ ID NOs: 2, 4, 6, 8 and 10.
1 shows the amino acid sequence of luCl protein.

FIG. 3 shows that the clone SaGluCl1 (short form “SC”) shows Xenopus (Xe)
nopus) shows the results of experiments expressed in oocytes. The measurement was performed with two microelectrode voltage clamp techniques and the membrane potential was maintained at 0 mV. Two current records are overlaid. The top bar shows the duration of application of glutamate and ivermectin phosphate.

FIG. 4A and FIG. 4B show that ivermectin (FIG. 4A) and nodulasporic acid expressed in CHO cells are assembled into homomultimers.
d) SaGluCl1 (short form “SC”) activated by (FIG. 4B). 100 nM ivermectin and 1 μM nodulasporic acid were added at 20 seconds and the current was measured.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a Schistocerca americana.
ricana) (grasshopper) isolated nucleic acid molecule (polynucleotide) encoding an invertebrate GluCl channel protein. The nucleic acid molecule of the present invention is substantially free of other nucleic acids. DN for most cloning purposes
A is the preferred nucleic acid.

The present invention is a novel Schistocerca americana (Schistocerca a)
(nucleic acid) (grasshopper) isolated nucleic acid molecule (polynucleotide) encoding mRNA expressing invertebrate GluCl channel protein
Regarding This DNA molecule comprises the nucleotide sequences disclosed herein as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9.

Isolation and identification of the SaGluCl nucleic acid molecule of the present invention was confirmed by degenerate PCR as described in detail in Example 1. "TA" Cloning Kit (Inv
itrogen, Inc. ) Is used to generate a PCR product of appropriate size for PCR2
． 1 cloned into a plasmid vector. Sequence analysis (ABI Prism
, PE Applied Biosystems) followed by radiolabelling of the selected PCR clone insert and using it as a probe with the poly (A) -enriched fraction from the RNA, Uni-ZAP vector (Stratagen).
e, Inc. The cDNA library prepared in () was screened. The sequence from the full-length cDNA clone was cloned from GCG Inc. Analyzed using the package. SaGluCl1 clone and SaGlu into a mammalian expression vector
Subcloning of Cl2 was performed by excising the entire insert from the UniZap pBS plasmid (EcoRI + XhoI) followed by Tet.
It was performed by ligation into Splice (Life Technologies Inc.). Invertebrate glutamate-gated chloride channel (GluCl
) Is associated with glycine and GABA-gated chloride channels and is distinct from excitatory glutamate receptors such as the NMDA or AMPA receptors. The first two members of the GluCl family have been subjected to a functional screen for receptors for the anthelmintic drug ivermectin, according to C. elega.
ns). Several additional GluCls are currently being cloned in other invertebrate species. However, GluC in vertebrates
There is still no evidence of a counterpart. This makes GluCl an attractive target for anthelmintics, insecticides, acaricides and the like. In fact, the specific GluCl
Modulators such as nodulasporic acid and its derivatives have an ideal safety profile. Because they are
This is because vertebrates lack toxicity based on the mechanism of action. The present invention provides, in part, 2 from the ventral ganglia of Grasshopper Cistocerca americana by reverse transcription polymerase chain reaction (RT-PCR) using degenerate oligonucleotides as reaction primers.
It concerns two novel GluCl clones (SaGluCl1 and GluCl2). Full-length SaGluCl1 cDNA clone (SEQ ID NOs: 1, 3, 5 and 7)
Sequence data for D. melanogaster D
rosGluClα and C. felis CfGluCl DNA
Shows homology to. Xenopus laev
is) Heterologous expression of a short form of SaGluCl1 in oocytes causes a vigorous L-glutamate-dependent chloride current. This channel is also activated by nodulasporic acid and ivermectin. Full length SaGluCl2
The sequence of the cDNA clone (SEQ ID NO: 9) shows moderate homology to various GluCl. Co-expression of SaGluCl2 and SaGluCl1 strongly diminished the response to the compound, suggesting a role for modulators of SaGluCl2. From previous work on other four-transmembrane ligand-gated channel receptors (acetylcholine, GABA receptors, etc.), most, if not all, native channels show that heteromultimers (ie, those containing at least two different subunits) , Pentamer). Sa
To demonstrate that GluCl1 and SaGluCl2 can co-assemble into functional channels, both clones were cloned in Xenopus oocytes and CH.
Co-expressed in O cells. No current was observed in either case when both clones were co-expressed. Therefore, under these heterologous expression conditions,
SaGluCl2 seems to have a dominant negative effect on SaGluCl1. Nevertheless, the data probably indicate that both subunits are highly expressed in the same neuron, presumably the third GluCl of the native GluCl.
The component will be unidentified. Alternatively, it remains possible that SaGluCl1 and SaGluCl2 are not expressed in common (neuronal) cells within the ganglion and cannot co-assemble into common functional channels. Regardless, as shown in FIG. 3 (Xenopus oocytes) and FIGS. 4A and 4B (CHO cells), expression of the short form of SaGluCl1 resulted in functional homozygous responses to glutamate, ivermectin and nodulasporic acid. Results in the formation of multimeric channels.

Thus, SaGluCl2, when co-expressed with the short form of SaGluCl1, prevents the formation of a functional channel that would otherwise be detectable by SaGluCl1 alone. Therefore, the SaGluCl2 protein is
Expressed to act as a regulator of uCl1. Both SaGluCl1 and SaGluCl2 are expressed in the same tissue, which shows GluCl activity according to electrophysiological recordings. Therefore, 1) SaGluCl1 and SaGluCl2 are not expressed in exactly the same cell, and / or 2
2.) Another SaGluCl form or an unrelated type of protein, such as that found in the TipE protein from Drosophila, which enhances in vitro expression of parasodium channels (Fen
g., et al., 1995, Cell 82: 1001-1011))). In addition, SaGluCl2 is a non-SaGluC
It has been shown that it may have a negative effect on the 14 transmembrane ligand-gated ion channel (4TMLGIC), but not on other types of ion channels. Thus, the SaGluCl protein, which is specifically associated with the four-transmembrane ligand-gated ion channel, can be classified as a potential broad-spectrum inhibitory subunit and this isolated cDNA Clones, related vectors, hosts, recombinant subcellular fractions and membranes and expressed and mature forms of SaGluCl2 are useful as important tools for drug discovery.

To this end, the present invention is directed, in part, to invertebrates such as nematodes (C. e.
legans)) or a vertebrate (eg mouse), characterized in that the gene encoding SaGluCl2 has been introduced into the germline of said animal. The purpose would be to inactivate one or several endogenous 4TMLGICs in the host and observe the biological effect. One such effect is 4TMLGIC
It may be acquired resistance to a drug that is an agonist (activator) of. 4TM
For a drug with a suspected but unproven mode of action (MOA) via LGIC:
Such SaGluCl2-bearing transgenic animals can be used to confirm such effects. Expression of the newly introduced gene encoding SaGluCl2 in the host can be constitutive or inducible, depending on the type of promoter used to drive that expression. Also, depending on the type of promoter used, it is possible to target SaGluCl2 expression to a given tissue or to generalize it. The same reasoning can be applied to in vitro expression in, for example, Xenopus oocytes. SaGlu
Co-expression of Cl2 with any other 4TMLGIC, including functional SaGluCl1 and non-locust forms, should interfere with any 4TMLGIC-mediated current. Xenopus oocytes can be injected with a mixed population of mRNA or RNA, eg, from the mammalian brain or from whole animals (eg, C. elegans). Then these oocytes, sometimes unknown M
Attach to OA drug. The use of SaGluCl2 results in suppression of the drug response, thus limiting the course of the study to 4TMLGIC and serves to identify various preferred modulators of 4TLMGIC.

Thus, heterologous expression of grasshopper GluCl channels, in particular SaGluCl1 alone, or co-expression of SaGluCl2 as described above, is a drug of compounds active against parasitic invertebrate species associated with animal and human health. It will enable a physical analysis. Such species include helminths, fleas, ticks and lice. Heterologous cell lines expressing these GluCl channels can be used to establish functional or binding assays to identify novel GluCl channel modulators that may be useful in the control of said species.

Grasshopper Gl shown in FIGS. 2A-B as 1LA and described as SEQ ID NO: 2
SAGluCl1LA (Sa1L in FIGS. 1A-F, which encodes the uCl protein).
The nucleotide sequence of the DNA molecule shown as A) and described as SEQ ID NO: 1 is as follows:

[0051]

[Chemical 5]

Grasshopper Gl shown as 1LC in Figures 2A-B and set forth as SEQ ID NO: 4
SAGluCl1LC (Sa1L in FIGS. 1A-F, which encodes the uCl protein.
The nucleotide sequence of the DNA molecule shown as C) and described as SEQ ID NO: 3 is as follows:

[0053]

[Chemical 6]

Grasshopper Gl shown as 1SA in Figures 2A-B and set forth as SEQ ID NO: 6
SAGluCl1SA (Sa1S in FIGS. 1A-F, which encodes the uCl protein).
The nucleotide sequence of the DNA molecule shown as A) and described as SEQ ID NO: 5 is as follows:

[0055]

[Chemical 7]

Grasshopper Gl shown as 1SC in Figures 2A-B and set forth as SEQ ID NO: 8
SAGluCl1SC (Sa1S in FIGS. 1A-F, which encodes the uCl protein).
The nucleotide sequence of the DNA molecule shown as C) and described as SEQ ID NO: 7 is as follows:

[0057]

[Chemical 8]

Grasshopper G, shown as “2” and depicted as SEQ ID NO: 10 in FIGS.
The nucleotide sequence of the DNA molecule encoding the luCl protein, shown as SaGluCl2 in FIGS. 1A-F and set forth as SEQ ID NO: 9, is as follows:

[0059]

[Chemical 9]

The above-described isolated DNA molecule shown in FIGS.
"GA" termination codon (nucleotides 1845-1847); SAGluCl1L (Sa1LC; SEQ ID NO: 3): 1879 nucleotides: start Met (nucleotides 306-308) and "T.
"GA" termination codon (nucleotides 1845-1847); SAGluCl1S (Sa1SA; SEQ ID NO: 5): 1776 nucleotides: start Met (nucleotides 326-328) and "T.
"GA" termination codon (nucleotides 1712-1713); SAGluCl1S (Sa1SC; SEQ ID NO: 7): 1776 nucleotides: start Met (nucleotides 326-328) and "T.
"GA" termination codon (nucleotides 1712-1713); SAGluCl12 (Sa2; SEQ ID NO: 9): 2074 nucleotides: start Met (nucleotides 41-43) and "TGA.
A termination codon (nucleotides 1382-1384).

For the “long” form of SaGluCl1, the “LA” cDNA (SEQ ID NO: 1
) Encodes a protein having a Glu residue at amino acid number 511,
The cDNA (SEQ ID NO: 3) encodes a protein having an Asp residue at amino acid number 511.

For the “short” form of SaGluCl1, the “SA” cDNA (SEQ ID NO: 5
) Encodes a protein having a Glu residue at amino acid number 460, the "SC
The cDNA (SEQ ID NO: 7) encodes a protein with an Asp residue at amino acid number 460.

The present invention also relates to biologically active fragments or mutants of SEQ ID NOs: 1, 3, 5, 7 and 9 which encode mRNAs expressing SaGluCl1 or SaGluCl2, respectively. Any such biologically active fragments and / or
Alternatively, the mutant is also a protein that at least substantially mimics the wild-type protein (including but not limited to the wild-type forms set forth in SEQ ID NOs: 2, 4, 6, 8 and / or 10). Or it will encode either of the protein fragments. Any such polynucleotides include nucleotide substitutions, deletions, additions, amino-terminal truncation errors and carboxy-terminal truncation errors, where these mutations express a diagnostic or therapeutic or prophylactic protein or protein fragment.
RNA, which may be useful in screening for agonists and / or antagonists of SaGluCl function), but is not necessarily limited thereto.

A preferred embodiment of this form of the invention is disclosed in Figures 1A-F, which show cDNA molecules encoding various forms of the SaGluCl channel protein.

Isolated nucleic acid molecules of the present invention include deoxyribonucleic acid molecules (DNA), such as genomic DNA and complementary DNA (cDNA) [which may be single-stranded (coding or non-coding) or double-stranded. , As well as synthetic DNA, such as synthetic single-stranded polynucleotides. In addition, the isolated nucleic acid molecule of the present invention may include a ribonucleic acid molecule (RNA).

Due to the degeneracy of the genetic code, for every amino acid except two, 2
These codons code for one particular amino acid. Therefore, SEQ ID NOS: 1, 3
SEQ ID NOS: 1, 3, 5, although significantly different from the nucleotide sequences of 5, 7, and 9
It allows the construction of SaGluCl protein-encoding synthetic DNA having a synthetic DNA nucleotide sequence that still encodes the same SaGluCl protein as 7 and 9. Such synthetic DNA is intended to be within the scope of the present invention. If it is desired to express such synthetic DNA in a particular host cell or organism, the codon usage of such synthetic DNA may be adjusted to reflect the codon usage of the particular host, and High levels of expression of the SaGluCl channel protein in the host can be brought about. That is, this duplication at various codons encoding a particular amino acid is included within the scope of the invention. Therefore, the present invention also relates to a DNA sequence encoding an RNA containing alternative codons which code for the final translation of the same amino acids shown below.

A = Ala = alanine: codons GCA, GCC, GCG, GCU C = Cys = cysteine: codons UGC, UGU D = Asp = aspartic acid: codon GAC, GAU E = Glu = glutamic acid: codons GAA, GAG F = Phe = phenylalanine: codons UUC, UUU G = Gly = glycine: codons GGA, GGC, GGG, GGU H = His = histidine: codon CAC, CAU I = Ile = isoleucine: codons AUA, AUC, AUU K = Lys = lysine: Codon AAA, AAG L = Leu = Leucine: Codons UUA, UUG, CUA, CUC, CUG, CU
UM = Met = methionine: codon AUG N = Asp = asparagine: codon AAC, AAU P = Pro = proline: codon CCA, CCC, CCG, CCU Q = Gln = glutamine: codon CAA, CAG R = Arg = arginine: codon AGA, AGG, CGA, CGC, CGG, C
GU S = Ser = serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T = Thr = threonine: codons ACA, ACC, ACG, ACU V = Val = valine: codons GUA, GUC, GUG, GUU W = Trp = tryptophan: codon UGG Y = Tyr = tyrosine: codons UAC, UAU

Thus, the present invention discloses codon redundancy, which can result in differences in DNA molecules expressing the same protein. For purposes of this specification, sequences that carry one or more replacement codons are defined as degenerate variants. Also included within the scope of the invention are mutations in the DNA sequence or translated protein that do not substantially alter the ultimate physical properties of the expressed protein. For example, leucine to valine, lysine to arginine, or glutamine to asparagine is replaced by
It will not cause a change in the functionality of the polypeptide.

It is known that the DNA sequence encoding a peptide can be modified to encode a peptide that has different properties than its naturally occurring peptide.
Methods of modifying a DNA sequence include, but are not limited to, site-directed mutagenesis. Specific examples of properties that are modified include, but are not limited to, changes in the affinity of the enzyme for the substrate or the receptor for the ligand.

“Identity” is a measure of the identity of nucleotide or amino acid sequences.
Generally, the sequences are aligned for the highest match. “Identity” itself has the art-recognized definition and can be calculated using published techniques. For example, Computational Molecular Bi
oology, Lesk, A .; M. Hen, Oxford University P
less, New York, 1988; Biocomputing: Info.
rmmatics and Genome Projects, Smith, D.M.
W. Edited by Academic Press, New York, 1993; Com.
putter Analysis of Sequence Data, Part
I, Griffin, A .; M. And Griffin, H .; G. Hen, Huma
na Press, New Jersey, 1994; Sequence An
alysis in Molecular Biology, von Hein
je, G.G. , Academic Press, 1987; and Sequence.
e Analysis Primer, Gribskov, M .; And Dev
reux, J .; Edited, M Stockton Press, New York, 1
See 991. There are numerous methods for determining the identity between two polynucleotide or polypeptide sequences, the term "identity" being well known to those skilled in the art (Carillo and Lipton, 1988, SIAM JA.
pplyed Math 48: 1073). Commonly used methods for measuring identity or similarity between two sequences include Guide to Huge.
Computers, Martin J. Bishop ed., Academic
Press, San Diego, 1994 and Carillo and Li.
pton, 1988, SIAM J Applied Math 48: 107.
3 but are not limited thereto. Methods to determine identity and similarity are codified in computer programs. A preferred computer program method for determining identity and similarity between two sequences includes the GCG program package (Devereux et al., 19
84, Nucleic Acids Research 12 (1): 387).
, BLASTN and FASTA (Altschul et al., 1990, J Mol.
． Biol. 215: 403), but is not limited thereto. For example, a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7 and / or 9 is Means that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that it may contain up to 5 point mutations or alternative nucleotides per 100 nucleotides of the 3, 5, 7 and / or 9 reference nucleotide sequence. To do. That is, in order to obtain a polynucleotide sequence having a nucleotide sequence which is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence must be deleted or replaced by another nucleotide. Or it is possible that a large number of nucleotides up to 5% of all the nucleotides in the reference sequence have been inserted in the reference sequence. These mutations or alternative nucleotide substitutions of the reference sequence may be present at the 5'or 3'terminal positions of the reference nucleotide sequence, or at any position between those terminal positions within the reference sequence. Can be interspersed individually or as one or more contiguous groups within the reference sequence. One cause of such "mutations" or alterations that give less than 100% identity can result from RNA editing. The process of RNA editing results in the modification of the mRNA molecule, so that use of the modified mRNA as a template to make a cloned cDNA can result in one or more nucleotide changes, which results in codon changes. May or may not result in. It is known that this RNA editing is catalyzed by RNA editorase. Such an RNA editorase is RNA adenosine deaminase, which converts adenosine residues to inosine residues, which tends to mimic cytosine residues. For this purpose, conversion of mRNA residues from A to I will result in an A to G transition within the coding and non-coding regions of the cloned cDNA (eg, Hanrahan et al., 1999, Annals New. Yo
rk Acad. Sci. 868: 51-66; for a review,
Bass, 1997, TIBS 22: 157-162). Similarly, a polypeptide having an amino acid sequence having at least 95% identity to the reference amino acid sequence of SEQ ID NO: 2, 4, 6, 8 and / or 10 is:
The amino acid sequence of the polypeptide is identical to the reference sequence, except that the polypeptide sequence may include modifications of up to 5 amino acids per 100 amino acids of the reference amino acids of SEQ ID NOs: 2, 4, 6, 8 and / or 10. Means that. That is, in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence have been deleted or replaced with another amino acid. It is possible, or it is possible that up to 5% of all amino acid residues in the reference sequence are inserted in the reference sequence. These modifications of the reference sequence may occur at amino or carboxy terminal positions of the reference amino acid sequence, or at any position between those terminal positions, individually in residues within the reference sequence. Or it can be interspersed as one or more contiguous groups within the reference sequence. Again, as mentioned above, RNA editing involves codons that give rise to expressed proteins that differ in "identity" from other proteins expressed from "non-RNA editing" transcripts that correspond directly to the open reading frame of the genomic sequence. It can make a difference.

The present invention also relates to recombinant vectors and recombinant hosts (both prokaryotic and eukaryotic) containing the substantially purified nucleic acid molecules disclosed throughout this specification. A nucleic acid molecule of the invention encoding a SaGluCl channel binds, in whole or in part, to another DNA molecule (ie, a DNA molecule to which the SaGluCl coding sequence is not naturally associated) to bind the respective SaGluCl channel protein. It may form a "recombinant DNA molecule" which encodes. SaGlu
In a vector containing a nucleic acid encoding Cl or a functional equivalent, the novel D
NA sequences can be inserted. These vectors can include DNA or RNA. DNA vectors are preferred for most cloning purposes. Typical vectors include plasmids, modified viruses, bacteriophages, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode SaGluCl channel proteins. It is well within the skill of one in the art to determine the appropriate vector for a particular gene transfer or other use.

The present invention includes DNA sequences that hybridize to SEQ ID NOs: 1, 3, 5, 7 and 9 under stringent conditions. For example, methods using conditions of high stringency include, but are not limited to: Prehybridization of filters containing DNA was performed with 6xSSC
Perform in a buffer containing 5 × Denhardt's solution and 100 μg / ml denatured salmon sperm DNA at 65 ° C. for 2 hours to overnight. Filters are hybridized for 12-48 hours at 65 ° C. in a prehybridization mixture containing 100 μg / ml denatured salmon sperm DNA and 5-20 × 10 ⁶ cpm of ³² P-labeled probe. The filters are washed for 1 hour at 37 ° C. in a solution containing 2 × SSC, 0.1% SDS. This is followed by 4 at 50 ° C. in 0.1 × SSC, 0.1% SDS.
Wash for 5 minutes and then autoradiograph. Other methods using high stringency conditions are 5x SSC, 5x Denhardt's solution, 50%
A hybridization step in formamide at 42 ° C. for 12 to 48 hours,
Or would include a wash step in 0.2 x SSPE, 0.2% SDS at 65 ° C for 30-60 minutes.

The reagents listed in the above methods for performing high stringency hybridization are well known in the art. Details of the composition of these reagents can be found, for example, in Sambrook et al., 1989, Molecular Cloning:
A Laboratory Manual, Cold Spring Harb
or Laboratory, Cold Spring Harbor, New
It is described in York. Other high stringency conditions that may be used besides those mentioned above are well known in the art.

The present invention also includes a substantially purified form of each SaG comprising the amino acid sequences disclosed in FIGS. 2A-B and set forth as SEQ ID NOs: 2, 4, 6, 8 and 10.
relates to luCl channel proteins. The disclosed SaGluCl protein contains an open reading frame of 513 (SEQ ID NOS: 2 and 4), 462 amino acids (SEQ ID NOS: 6 and 8) and 447 (SEQ ID NO: 10) amino acids shown in Figures 2A-B and below. .

[0075]

[Chemical 10]

The present invention also includes (but is not necessarily limited to) amino acid substitutions, deletions, additions, amino-terminal truncation errors and carboxy-terminal truncation errors.
These mutations confer diagnostic or therapeutic or prophylactic proteins or protein fragments and may be useful in screening for agonists and / or antagonists of the function of SaGluCl), SEQ ID NOs: 2, 4, 6, 8 and 10. It relates to biologically active fragments and / or mutants of the SaGluCl protein comprising the described amino acid sequence.

Another preferred embodiment of the present invention is a recombinant containing a DNA expression vector comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7 and / or 9 and expressing the respective SaGluCl precursor protein. It relates to a substantially purified, fully processed GluCl channel protein obtained from a host cell. It is particularly preferred that the recombinant host cell is a eukaryotic host cell line such as a mammalian cell line or a Xenopus oocyte, as described above.

As with many proteins, it is possible to modify many of the amino acids of the SaGluCl channel protein and still retain substantially the same biological activity as the wild type protein. Accordingly, the present invention includes modified SaGluCl polypeptides that have amino acid deletions, additions or substitutions, but still retain substantially the same biological activity as their respective SaGluCl. It is generally accepted that a single amino acid substitution does not normally alter the biological activity of the protein (eg, Molecular Biology of the Gene,
Watson et al., 1987, 4th edition, The Benjamin / Cummin.
gs Publishing Co. , Inc. , P. 226; and Cunn
ingham & Wells, 1989, Science 244: 1081.
-1085). Therefore, the present invention provides SEQ ID NOs: 2, 4, 6, 8
And / or polypeptides with one amino acid substitution at 10 that still retain substantially the same biological activity as the corresponding SaGluCl protein. The present invention is also a polypeptide having 2 or more amino acid substitutions in SEQ ID NOs: 2, 4, 6, 8 and / or 10 which still retain substantially the same biological activity as the corresponding SaGluCl protein. Contains a polypeptide. In particular, the invention includes embodiments in which the substitution is a conservative substitution.

Those skilled in the art will also recognize that polypeptides that are functional equivalents of SaGluCl with changes from the SaGluCl amino acid sequence that are small deletions or insertions of amino acids also follow the same guidelines (ie, SaGluCl and related proteins). Will minimize the differences in the amino acid sequence between). Small deletions or insertions generally range from about 1-5 amino acids. The effect of such small deletions or insertions on the biological activity of the modified SaGluCl polypeptide can be by producing the polypeptide synthetically or by making the necessary changes in the SaGluCl encoding DNA and then combining the DNA. It can be easily assayed by alternatively expressing and assaying the protein produced by the recombinant expression.

The present invention also includes a truncated error form of SaGluCl containing a region containing the active site of the enzyme. Such truncation error proteins are useful in various assays described herein, and for crystallization studies and for structure-activity relationship studies.

The present invention also provides crude or substantial cells from recombinant host cells (both prokaryotic and eukaryotic, as well as stably and transiently transformed cells) containing the nucleic acid molecules of the invention. Subcellular membrane fraction purified to. These recombinant host cells express SaGluCl or a functional equivalent that is post-translationally associated with the cell membrane in a biologically active form. These subcellular membrane fractions have Sa levels at levels substantially above endogenous levels.
It includes wild-type or mutant forms of GluCl and thus would be useful in assays for selecting modulators of SaGluCl proteins or channels. Thus, a particular use for such subcellular membranes is to express SaGluCl in the recombinant cells and then to isolate and substantially purify the membrane from other cellular components, then to Use in assays to select modulators such as agonists or antagonists of biologically active channels containing one or more of the proteins disclosed herein. Therefore, another preferred embodiment of the present invention comprises a complete open reading frame as set forth in SEQ ID NOs: 1, 3, 5, 7 and / or 9 to express it appropriately to functionalize each SaGluCl channel. It relates to a substantially purified membrane preparation obtained from a recombinant host cell transformed or transfected with a DNA expression vector giving a processed form. The recombinant host cell is as described above,
Particularly preferred is a eukaryotic host cell line such as a mammalian cell line or a Xenopus oocyte.

The present invention also relates to an isolated nucleic acid molecule that is a fusion construct that expresses a fusion protein useful in an assay to identify compounds that modulate wild-type SaGluCl activity and that produces an antibody to SaGluCl. In a preferred embodiment of this aspect of the invention, glutathione S-transferase (GST) -Sa.
Includes, but is not limited to, GluCl fusion constructs. Recombinant G
The ST-SaGluCl fusion protein is a baculovirus expression vector (pA
cG2T, Pharmingen) using Spodoptera frugiperda (S
podoptera frugiperda (Sf21) insect cells (Invi
It can be expressed in various expression systems such as trogen). Another aspect is SaGluC linked to various markers including (but not limited to) GFP (Green Fluorescent Protein), MYC epitope and GST.
1 and / or SaGlu-2 fusion constructs. Again, any such fusion construct can be expressed in the cell line of interest and used to screen for one or more modulators of the SaGluCl protein disclosed herein.

A preferred embodiment for screening for modulators of SaGluCl channel activity is that the DNA molecules of the invention are Xenopus.
laevis) is an expression system for an electrophysiology-based assay to measure the activity of glutamate-gated chloride channels, including injection into oocytes. Xenopus in the study of ion channel activity
The general use of oocytes is known in the art (Dascal, 1
987, Crit. Rev. Biochem. 22: 317-317; Lest
er, 1988, Science 241: 1057-1063; see also Met.
hods of Enzymology, Vol. 207, 1992, Ch. 1
4-25, edited by Rudy and Iverson, Academic Press,
Inc. , New York). In part of the present invention, an improved method for measuring agonist and / or antagonist modulation and channel activity is disclosed, which is several times more sensitive than previously disclosed methods. DNA encoding a gated channel, the activity of which and the response of the channel to various regulatory factors can be measured, m
Nucleic acid material, including but not limited to RNA or cRNA,
Inject into Xenopus oocytes. Ion channel activity is measured by using a holding potential (preferably about 0 mV) that is more positive than the reversal potential of chloride (ie, greater than -30 mV). This modification in assay measurement conditions resulted in a 10-fold increase in assay sensitivity over modulation by ivermectin phosphate. Thus, this improved assay will allow screening and selection for compounds that modulate GluCl activity at levels previously thought to be undetectable. This method is outlined in the examples. It will be apparent to those skilled in the art that this method can be used in a variety of ion channel assay assays, particularly assays that measure glutamate-dependent activity in eukaryotic cells such as Xenopus oocytes. Caenorhabditis
elegans), Drosophila mel
onaster, cat flea (Ctenocephalides feli)
s) invertebrate glutamate-gated chloride channels, including but not limited to glutamate-gated channels and the S. americana glutamate-gated channel proteins disclosed herein, It is particularly preferred for use in an assay to screen and select for compounds that modulate the activity of these channels. Levels of SaGluCl protein in host cells are quantified by immunoaffinity and / or ligand affinity techniques. By measuring the amount of radioactive glutamate or ivermectin binding to the cell membrane, SaGl
Cells expressing uCl can be assayed for the number of GluCl molecules expressed. SaGluCl-specific affinity beads or SaGluC
l-specific antibodies can be used, for example, with ³⁵ S-methionine labeled or unlabeled SaGl.
uCl protein is isolated. Labeled SaGluCl protein is SDS-PA.
Analyze by GE. Unlabeled SaGluCl protein is detected by Western blotting, ELISA or RIA assay using SaGluCl specific antibodies.

Any of a variety of methods can be used to clone SaGluCl. These methods include, but are not limited to: (1) RACE PCR cloning technology (Frohman et al.,
1988, Proc. Natl. Acad. Sci. 85: 8998-9002
). 5'and / or 3'RACE can be performed to obtain the full length cDNA sequence. This method involves the use of gene-specific oligonucleotide primers for PCR amplification of SaGluCl cDNA. These gene-specific primers are designed through the identification of EST (expressing tag sequence) nucleotide sequences identified by searching a large number of publicly available nucleic acid and protein databases. (2) SaGluCl in a suitable expression vector system
Direct functional expression of SaGluCl cDNA after construction of the containing cDNA library. (3) Screening of SaGluCl-containing cDNA libraries constructed in bacteriophage or plasmid shuttle vectors with labeled degenerate oligonucleotide probes designed from the amino acid sequence of SaGluCl protein. (4) SaGluCl constructed in bacteriophage or plasmid shuttle vector with partial cDNA encoding SaGluCl protein
Screening of the contained cDNA library. This partial cDNA is SaGlu.
Obtained by specific PCR amplification of SaGluCl DNA fragments by designing degenerate oligonucleotide primers from amino acid sequences known for other kinases related to the Cl protein. (5) SaGluCl constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA or oligonucleotide having homology to mammalian SaGluCl protein
Screening of the contained cDNA library. This method may also involve the use of gene-specific oligonucleotide primers for PCR amplification of SaGluCl cDNA identified as EST as described above. (6) Design of 5'and 3'gene-specific oligonucleotides by using SEQ ID NO: 1, 3, 5, 7 or 9 as template. As a result, the full-length cDNA was converted to known RACE.
Of the same known RA
The complete nucleotide sequence encoding SaGluCl was generated by CE technology and isolated and isolated from a portion of the coding region used as a probe to screen one of the many types of cDNA and / or genomic libraries. It is possible to isolate the long form.

It will be readily appreciated by those skilled in the art that other types of libraries and libraries constructed from other cell types or species types may be useful for the isolation of SaGluCl encoding DNA or SaGluCl homologs. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells.

It will be readily appreciated by those skilled in the art that suitable cDNA libraries can be prepared from cells or cell lines that have SaGluCl activity. The selection of cells or cell lines for use in preparing a cDNA library for the isolation of a SaGluCl-encoding cDNA first involves measuring cell-associated SaGluCl activity, which is known for such purposes. Can be performed using any of the
It can be done by

Preparation of a cDNA library can be performed by standard techniques well known in the art. Well-known cDNA library construction techniques are described, for example, in Sambrook et al., 1989, Molecular Cloning: A.
Laboratory Manual; Cold Spring Harbor
Laboratory, Cold Spring Harbor, New Y
ork. The complementary DNA library is Clontec.
h Laboratories, Inc. And Stratagene are included (
It is available from a number of commercial sources (without limitation).

Those of skill in the art will readily recognize that the DNA encoding SaGluCl can also be isolated from a suitable genomic DNA library. Construction of a genomic DNA library can be done by standard techniques well known in the art. A well-known technique for constructing a genomic DNA library is Sambrook et al.
(Above). Probes based on the SaGluCl nucleotide sequences disclosed herein can be used to prepare genomic libraries (especially P1 artificial chromosome vectors) from which SaGluCl-containing genomic clones can be isolated. Methods for preparing such libraries are known in the art (Ioannno).
u et al., 1994, Nature Genet. 6: 84-89).

In order to clone the SaGluCl gene by one of these preferred methods, the amino acid or DNA sequence of SaGluCl or a homologous protein may be required. To achieve this, each SaGluCl channel protein can be purified and the partial amino acid sequence determined by an automated sequenator. Although it is not necessary to determine the entire amino acid sequence,
For PCR amplification of the partial SaGluCl DNA fragment, it is possible to determine the linear sequence of two regions of 6-8 amino acids. Once the appropriate amino acid sequences have been identified, DNA sequences capable of encoding them are synthesized. Due to the degeneracy of the genetic code, more than one codon may be used to encode one particular amino acid. Thus, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set is SaGluCl
Will be the same as the array. However, the other members of the set could hybridize to SaGluCl DNA even in the presence of mismatched DNA oligonucleotides. The mismatched DNA oligonucleotide is still capable of hybridizing to SaGluCl DNA to a sufficient extent to allow identification and isolation of SaGluCl encoding DNA. Alternatively, the nucleotide sequence of a region of the expressed sequence can be identified by searching one or more available genomic databases. cDNA library or c
Gene-specific primers can be used to perform PCR amplification of a cDNA of interest from a DNA population. As mentioned above, suitable nucleotide sequences for use in PCR-based methods can be obtained from SEQ ID NOs: 1, 3, 5, 7 or 9 and used to duplicate 5'and 3 'Isolate the RACE product to generate the full-length sequence encoding SaGluCl, or
A portion of the nucleotide sequence encoding aGluCl is isolated and used as a probe to screen one or more of a cDNA or genome-based library to isolate a full-length sequence encoding a SaGluCl or SaGluCl-like protein. It is possible.

The present invention also provides a vector containing the SaGluCl gene, a host cell containing the vector, and the SaGluCl gene introduced into the host cell to produce SaGluCl.
A method for producing a substantially pure SaGluCl protein comprising culturing the host cell under suitable conditions so that Cl is produced. The SaGluCl thus produced can be recovered from the host cell by a conventional method. Accordingly, the present invention also provides methods for expressing SaGluCl proteins or bioequivalents (as described herein), assays using these gene products, recombinant hosts containing DNA constructs expressing these proteins. Cells, and SaGl
It relates to compounds identified by these assays that act as agonists or antagonists of uCl activity.

The cloned SaGluCl cDNA obtained by the method described above is used as an expression vector (eg pcDN) containing an appropriate promoter and other appropriate transcriptional regulatory elements.
A3. neo, pcDNA3.1, pCR2.1, pBlueBacHis2 or pLITMUS28) for recombinant expression by molecular cloning.
It can be introduced into prokaryotic or eukaryotic host cells to produce recombinant SaGluCl. The expression vector is, in the present invention, a cloned D in a suitable host.
It is defined as the DNA sequence required for the transcription of NA and the translation of their mRNA. Such a vector can be used to express eukaryotic DNA in various hosts such as bacteria, cyanobacteria, plant cells, insect cells, animal cells and the like. Specially designed vectors allow a DNA shuttle between hosts such as bacteria-yeast or bacteria-animal cells. A properly constructed expression vector should contain an origin of replication for autonomous replication in the host cell, a selectable marker, a number of useful restriction enzyme sites, a potentially high copy number and an active promoter. . A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. Strong promoters are those that result in high frequency synthesis of mRNA. To determine SaGluCl cDNA sequences that give optimal levels of SaGluCl, cDNA molecules can be constructed, including but not limited to: cDNA containing the full-length open reading frame of SaGluCl. Various constructs containing fragments, and portions of cDNA encoding only specific domains of the protein or rearrangement domains of the protein. All constructs can be designed to contain all or part of the 5'and / or 3'untranslated regions of the SaGluCl cDNA, or none at all. SaGluCl expression levels and activity can be measured after introduction of these constructs, alone or in combination, into appropriate host cells. After determining the SaGluCl cDNA cassette that gives optimal expression in the transient assay, the SaGluCl cDNA construct is used to contain expression vectors for mammalian cells, plant cells, insect cells, oocytes, bacteria and yeast cells. Various expression vectors (including but not limited to recombinant viruses)
To introduce. Techniques for such manipulations are described in Sambrook et al. (Supra) and are well known and available to those of skill in the art. Accordingly, another aspect of the invention includes a host cell engineered to contain and / or express a DNA sequence encoding SaGluCl. SaGlu in recombinant host cells
To express Cl, an expression vector containing DNA encoding a SaGluCl-like protein can be used. Such recombinant host cells can be cultured under suitable conditions to produce SaGluCl or bioisostere. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids or viruses. Commercially available mammalian expression vectors that may be suitable for expression of recombinant SaGluCl include pcDNA3. neo (Inv
itrogen), pcDNA3.1 (Invitrogen), pCI-ne
o (Promega), pLITMUS28, pLITMUS29, pLITM
US38 and pLITMUS39 (New England Biolab)
s), pcDNAI, pcDNAIamp (Invitrogen), pcDN
A3 (Invitrogen), pMC1neo (Stratagene), p
XT1 (Stratagene), pSG5 (Stratagene), EBO
-PSV2-neo (ATCC 37593), pBPV-1 (8-2) (AT
CC 37110), pdBPV-MMTneo (342-12) (ATCC
37224), pRSVgpt (ATCC 37199), pRSVneo (A
TCC 37198), pSV2-dhfr (ATCC 37146), pUC
Tag (ATCC 37460) and 1ZD35 (ATCC 37565), but are not limited thereto. In addition, recombinant Sa
A variety of bacterial expression vectors can be used to express GluCl. Commercially available bacterial expression vectors that may be suitable for expression of recombinant SaGluCl include pCR2.1 (Invitrogen), pET11.
a (Novagen), lambda gt11 (Invitrogen) and pKK
223-3 (Pharmacia), but is not limited thereto. In addition, various fungal cell expression vectors can be used to express recombinant SaGluCl in fungal cells. Commercially available fungal cell expression vectors that may be suitable for expression of recombinant SaGluCl include pY
ES2 (Invitrogen) and Pichia expression vector (
Invitrogen), but is not limited thereto. In addition, various insect cell expression vectors can be used to express the recombinant protein in insect cells. Commercially available insect cell expression vectors that may be suitable for recombinant expression of SaGluCl include pBlueBacII.
I and pBlueBacHis2 (Invitrogen), and pAcG
2T (Pharmingen) is included, but the present invention is not limited thereto.

Recombinant host cells can be prokaryotic or eukaryotic, including bacteria such as E. coli, fungal cells such as yeast, cows, pigs, monkeys and rods. Includes mammalian cells, including (but not limited to) cell lines derived from rodents, and insect cells, including (but not limited to) cell lines derived from Drosophila and Bombyx mori. However, it is not limited thereto. For example, one insect expression system is Spodoptera with the baculovirus expression vector (pAcG2T, Pharmingen).
The method uses Sfludoptera frugiperda (Sf21) insect cells (Invitrogen). Further, suitably a is Ur mammalian species are commercially available, L cells ^{L-M (TK -) (} ATCC CCL
1.3), L cell LM (ATCC CCL 1.2), Saos-2 (ATC
C HTB-85), 293 (ATCC CRL 1573), Raji (AT
CC CCL 86), CV-1 (ATCC CCL 70), COS-1 (A
TCC CRL 1650), COS-7 (ATCC CRL 1651), C
HO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92),
NIH / 3T3 (ATCC CRL 1658), HeLa (ATCC CCL
2), C127I (ATCC CRL 1616), BS-C-1 (ATCC
CCL 26), MRC-5 (ATCC CCL 171) and CPAE (
ATCC CCL 209), but is not limited thereto.

Xenopus (Xenop) for expressing the SaGluCl gene of interest
us) As described above with respect to the use of oocytes, the present invention relates to a method of screening for compounds that regulate the expression of DNA or RNA encoding SaGluCl protein. Compounds that regulate these activities can be DNA, RNA, peptides, proteins or non-proteinaceous organic molecules. The compound is SaGlu
It may be regulated by enhancing or diminishing the expression of DNA or RNA encoding Cl or the function of channels based on SaGluCl. Compounds that modulate the expression of SaGluCl-encoding DNA or RNA or its biological function can be detected by a variety of assays. The assay is a simple "yes / no" (yes / n) to determine if there is a change in expression or function.
o) ”assay. The assay can be made quantitative by comparing the expression or function of the test sample to the level of expression or function in a standard sample. Kits containing SaGluCl, antibodies to SaGluCl or modified SaGluCl can be prepared by known methods for such uses.

Using the DNA molecules, RNA molecules, recombinant proteins and antibodies of the invention,
SaGluCl can be screened and its level measured. The recombinant proteins, DNA molecules, RNA molecules and antibodies are useful in formulating kits suitable for the detection and typing of SaGluCl. Such a kit
It will include a compartmentalized carrier suitable for hermetically containing at least one container. The carrier may further comprise a reagent (eg, recombinant SaGluCl, or anti-SaGluC suitable for detection of SaGluCl).
1 antibody). Further, the carrier may contain a detection means such as a labeled antigen or an enzyme substrate.

The assays described herein can be performed using cells transiently or stably transfected with SaGluCl. The expression vector can be introduced into the host cell by any one of a number of techniques, including but not limited to transformation, transfection, protoplast fusion and electroporation. Transfection is meant to include any method known in the art for introducing SaGluCl into test cells. For example, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection of retroviral constructs containing SaGluCl, and electroporation.
The expression vector-containing cells are individually analyzed to determine if they produce SaGluCl protein. Identification of cells expressing SaGluCl can be accomplished by several means, including immunoreactivity with anti-SaGluCl antibodies, labeled ligand binding,
This can be done by the presence of, but not limited to, host cell-associated SaGluCl activity via ligand binding.

The specificity of binding of compounds showing affinity for SaGluCl is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells. Screening for compounds that bind SaGluCl, or for compounds that inhibit the binding of known radiolabeled ligands of SaGluCl to these cells or membranes prepared from these cells, and expression of the cloned receptor was performed against SaGluCl. And provides an efficient method for the rapid selection of compounds with high affinity. Such a ligand need not be radiolabeled, but can be a non-isotopic compound that can be used to displace the bound radiolabeled compound or can be used as an activator in a functional assay. The compound identified by the above method is considered to be an agonist or antagonist of SaGluCl and may be a peptide, protein or non-proteinaceous organic molecule.

Accordingly, the present invention relates to compounds that regulate the expression of DNA or RNA encoding SaGluCl protein and methods of screening for compounds that affect the function of said SaGluCl protein. Methods for identifying agonists and antagonists of other receptors are well known in the art,
It can be applied to identify agonists and antagonists of SaGluCl. For example, Cascieri et al. (1992, Molec. Pharmacol.
． 41: 1096-1099) describe a method for identifying substances that inhibit agonist binding to rat neurokinin receptors and are potential agonists or antagonists of neurokinin receptors. The method comprises transfecting COS cells with an expression vector containing a rat neurokinin receptor, culturing the transfected cells for a time sufficient to allow expression of the neurokinin receptor, Fected cells are harvested and resuspended in an assay buffer containing a known radiolabeled agonist of the neurokinin receptor in the presence or absence of the substance, followed by emission of the neurokinin receptor. Measuring the binding of a known agonist labeled to the neurokinin receptor. A substance is a potential agonist or antagonist of the neurokinin receptor when the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance. When binding of a substance such as an agonist or an antagonist is measured, such binding can be measured by using a labeled substance or agonist. The substance or agonist can be labeled (eg, radioactive, fluorescent, enzymatic) by any convenient method known in the art.

Thus, the specificity of binding of compounds with affinity for SaGluCl is demonstrated by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells. SaGl
Screening for compounds that bind uCl, or for compounds that inhibit the binding of radiolabeled known ligands of SaGluCl (eg, glutamic acid, ivermectin or nodulasporic acid) to these cells or membranes prepared from these cells, and The expression of the cloned receptor is SaGlu
It provides an efficient method for the rapid selection of compounds with high affinity for Cl. Such a ligand need not be radiolabeled, but can be a non-isotopic compound that can be used to displace the bound radiolabeled compound or can be used as an activator in a functional assay. The compound identified by the above method is considered to be an agonist or antagonist of SaGluCl and may be a peptide, protein or non-proteinaceous organic molecule. Compounds, as described elsewhere herein, are characterized by enhancing or attenuating the expression of DNA or RNA encoding SaGluCl or Sa
It can be regulated by acting as an agonist or antagonist of the GluCl receptor protein. Again, these compounds that modulate the expression of SaGluCl-encoding DNA or RNA or their biological function can be detected by various assays. The assay is a simple "yes / no" to determine if there is a change in expression or function.
It can be an assay. The assay can be made quantitative by comparing the expression or function of the test sample to the level of expression or function in a standard sample.

To this end, the present invention relates, in part, to SaGluCl1 and / or S
A method for identifying a substance that regulates aGluCl2 receptor activity, comprising: (a) SaGluCl1 and / or SaGluCl2 receptor protein (
The receptor protein comprises a test substance in the presence and absence of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8 and / or 10), and (b) the SaGluCl1 and / or SaGluCl2 receptor It relates to a method comprising measuring and comparing the effect of said test substance in the presence and absence of protein.

Also, some specific screening or selection assays are presented to show the various types of screening or selection assays that can be used by those of skill in the art with expression vectors that direct the expression of the SaGluCl1 and / or SaGluCl2 receptor proteins and additional SaGluCl. Embodiments are disclosed in the present invention. Methods for identifying agonists and antagonists of other receptors are well known in the art and can be applied to identify agonists and antagonists of SaGluCl1 and / or SaGluCl2. Therefore, these embodiments are described by way of illustration and not limitation. To this end, the present invention provides a SaG
Includes assays that can identify modulators of luCl1 and / or SaGluCl2 (eg, agonists and antagonists). Accordingly, the present invention provides a method for determining whether a substance is a potential agonist or antagonist of SaGluCl1 and / or SaGluCl2, which comprises (a) expression of SaGluCl1 and / or SaGluCl2 in a cell. Transfecting or transforming the cells with a directed expression vector, and (b) a known G that co-assembles with SaGluCl1 and / or SaGluCl2.
transfecting or transforming the cells with a second expression vector directing the expression of the luCl subunit to obtain test cells, (c) allowing expression of SaGluCl1 and / or SaGluCl2 and generation of functional channels For a sufficient period of time to: (d) SaGluCl1 and / or Sa in the presence and absence of the substance
Exposing the cells to a labeled ligand of GluCl2, and (e) measuring the binding of the labeled ligand to the SaGluCl1 and / or SaGluCl2 channels, the amount of binding of the labeled ligand in the absence of the substance. Less in the presence of the substance, the method is characterized in that the substance is a potential agonist or antagonist of SaGluCl1 and / or SaGluCl2.

The conditions under which step (d) of the method is carried out are those typically used in the art for the study of protein-ligand interactions (eg physiological pH; eg P
Salt conditions typical for commonly used buffers such as BS or in tissue culture medium; temperatures of about 4 ° C to about 55 ° C). The test cells can be harvested and resuspended in the presence of the substance and the labeled ligand. In a modification of the above method, instead of recovering and resuspending the cells, a radiolabelled agonist and the substance are contacted with the cells while the cells are attached to a substrate (eg, tissue culture plate). So that step (d) is modified.

The present invention also provides that certain substances are SaGluCl1 and / or SaGluCl2.
Whether or not the substance can bind to SaGluCl1 and / or Sa
A potential agonist or antagonist of GluCl2 channel activation), comprising: (a) transfecting the cell with an expression vector that directs expression of SaGluCl1 and / or SaGluCl2 in the cell. Test cells, which have been transfected or transformed with a second expression vector that directs the expression of at least one other known GluCl subunit that has been transfected or transfected with (b) SaGluCl1 and / or SaGluCl2. (C) exposing the test cells to the substance, (d) determining the amount of binding of the substance to SaGluCl1 and / or SaGluCl2, and (e) SaGluCl1 and / or SaGluCl2 in the test cells.
Comparing the amount of binding of said substance to a control cell not transfected with SaGluCl1 and / or SaGluCl2, wherein the amount of binding of said substance is The method is characterized in that the substance is capable of binding SaGluCl1 and / or SaGluCl2 if it is more abundant in the test cell compared to the cell. Then, for example, promiscuous as described below
Functional assays, such as assays involving the use of G proteins, can be used to determine whether the substance is in fact an agonist or antagonist.

The conditions under which step (c) of the method is carried out are those typically used in the art for the study of protein-ligand interactions (eg physiological pH; eg P
Salt conditions typical for commonly used buffers such as BS or in tissue culture medium; temperatures of about 4 ° C to about 55 ° C). The test cells can be harvested and resuspended in the presence of the substance.

Expression of SaGluCl DNA can also be performed using in vitro obtained synthetic mRNA. Synthetic mRNAs can be efficiently translated in a variety of cell-free systems, including but not limited to wheat germ extract and reticulocyte extract, and frog oocytes. Microinjection into frog oocytes is preferred, as it can be efficiently translated in a cell-based system that contains microinjection into.

After expressing SaGluCl in the host cell, the SaGluCl protein can be recovered to obtain the active form of the SaGluCl protein. Several SaGluCl protein purification methods are available and suitable for use. Recombinant S from cell lysates and extracts by various combinations of sole fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography or alone.
The aGluCl protein can be purified. In addition, full-length SaGluCl
Recombinant SaGluCl protein can be separated from other cellular proteins by the use of immunoaffinity columns made with monoclonal or polyclonal antibodies specific for the protein or polypeptide fragments of the SaGluCl protein.

The polyclonal or monoclonal antibody may be SaGluCl or SaGluCl1 or SaGl as set forth in SEQ ID NOs: 2, 4, 6, 8 and / or 10.
It can be raised against synthetic peptides (usually about 9 to about 25 amino acids in length) derived from a portion of uCl2. Monospecific antibodies against SaGluCl are Sa
Purified from mammalian antisera containing antibodies reactive to GluCl, or alternatively Kohler and Milstein (1975, Nature 256:
495-497) as a monoclonal antibody reactive with SaGluCl. Monospecific antibody as used in the present invention is defined as a plurality of antibody species or a single antibody species having uniform binding properties for SaGluCl. Homogenous binding, as used in the present invention, depends on the specific antigen or epitope (eg, SaG described above).
(associated with luCl) means that the antibody species can bind. Human SaGlu
The Cl-specific antibody is used for the animals such as mice, rats, guinea pigs, rabbits, goats and horses in the presence or absence of an immunoadjuvant in an appropriate concentration of SaGluC.
It is produced by immunization with a synthetic peptide made from l protein or part of SaGluCl.

Pre-immune serum is collected prior to the first immunization. Each animal receives from about 0.1 mg to about 1000 mg of SaGluCl protein with an acceptable immune adjuvant. Such acceptable adjuvants include Freund's complete, Freund's incomplete, alum precipitate, tRNA and Corynebacterium parvum (Cory.
water-in-oil emulsions containing nebacterium parvum), but are not limited thereto. The primary immunization preferably consists of administering SaGluCl protein or its peptide fragments in Freund's complete adjuvant to multiple sites subcutaneously (SC), intraperitoneally (IP) or both. Blood is drawn from each animal at regular intervals (preferably weekly) to measure antibody titers. After the first immunization, the animals may or may not receive booster injections. Animals receiving booster injections generally received an equal volume of S in Freund's incomplete adjuvant.
aGluCl is given by the same route. Booster injections are given at approximately 3 week intervals until maximum titers are obtained. About 7 days after each booster immunization, or almost weekly after a single immunization, the animals are bled, serum is collected, and aliquots are stored at about -20 ° C.

A monoclonal antibody (mAb) reactive with SaGluCl was used as an inbred mouse,
It is preferably prepared by immunizing Balb / c with the SaGluCl protein. The mice are immunized by the IP or SC route with about 1 mg to about 100 mg, preferably about 10 mg, of SaGluCl protein in about 0.5 ml of buffer or saline in an equal volume of the acceptable adjuvant. Freund's complete adjuvant is preferred. The mice are primed on day 0 and rested for about 3 to about 30 weeks. Immunized mice were given a booster immunization of about 1 to about 100 mg SaGluCl intravenously (I) in a buffer solution such as phosphate buffered saline.
V) Do one or more times by route. Lymphocytes, preferably splenic lymphocytes, are obtained from antibody-positive mice by removing the spleen from the immunized mice by standard methods known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with a suitable fusion partner (preferably myeloma cells) under conditions that allow the formation of stable hybridomas. The fusion partner is mouse myeloma P3 / NS1 / A
It may include, but is not limited to, g4-1, MPC-11, S-194 and Sp2 / 0. Of these, Sp2 / 0 is preferred. The antibody-producing cells and myeloma cells are fused in polyethylene glycol having a molecular weight of about 1000 at a concentration of about 30% to about 50%. Fused hybridoma cells are selected by growth in Dulbecco's modified Eagle's basal medium (DMEM) supplemented with hypoxanthine, thymidine and aminopterin by methods known in the art. Supernatant fluid was collected from growth positive wells at about 14, 18 and 21 days, and the S
Screen for antibody production by immunoassays such as solid phase immunoradioassay (SPIRA) using aGluCl. The culture fluid is also tested in an Ouchterlony precipitation assay to determine the isotype of the mAb.
Hybridoma cells from antibody positive wells were transferred to MacPherson, 1973.
, Soft Agar Technologies, Tissue Culture
Methods and Applications, Kruse and Pa
Cloning is performed by a technique such as the soft agar technique of ederson by Terson, Academic Press.

Pristane-sensitized Balb / c mice were exposed to about 2 × 10 ⁶ to about 6 about 4 days after sensitization.
Monoclonal antibodies are produced in vivo by injecting × 10 ⁶ hybridoma cells (approximately 0.5 ml / mouse). Ascites fluid is collected about 8-12 days after cell transfer and the monoclonal antibody is purified by techniques known in the art.

In vitro production of anti-SaGluCl mAb is performed by growing the hybridoma in DMEM containing about 2% fetal bovine serum to obtain sufficient quantities of the specific mAb. The mAb is purified by techniques known in the art.

Antibody titers in ascites or hybridoma cultures can be assayed for a variety of techniques including, but not limited to, precipitation, passive agglutination, enzyme linked antibody immunosorbent (ELISA) and radioimmunoassay (RIA) techniques. It is measured by a serological or immunological assay. A similar assay is used to detect the presence of SaGluCl in body fluids or tissue and cell extracts.

It will be readily appreciated by those skilled in the art that the methods for producing monospecific antibodies can be used to produce antibodies specific for the SaGluCl peptide fragment or the respective full length SaGluCl.

For example, N- so that the antibody forms a covalent bond with the agarose gel bead support.
Aff, a gel support pre-activated with hydroxysuccinimide ester
By adding the antibody to igel-10 (Biorad), SaGluCl
Create an antibody affinity column. The antibody is then attached to the gel via an amide bond with a spacer arm. Then, remove the remaining activated ester by 1M.
Quench with ethanolamine HCl (pH 8). The column was washed with water, then 0.
Wash with 23 M glycine HCl (pH 2.6) to remove unconjugated antibody or foreign protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and cell culture supernatant or cell extract containing full-length SaGluCl or SaGluCl protein fragments is slowly passed through the column. Then, until the optical density _{(A 280)} falls to background, then washing the column with phosphate buffered saline, then 0.23M glycine -HCl the protein (pH 2.6
) To elute. The purified SaGluCl protein is then dialyzed against phosphate buffered saline.

The present invention also provides SaGluCl by obtaining cells for culturing in vitro.
1, non-human transgenic animals useful for studying the ability of various compounds to act in vitro as modulators of SaGluCl2 or any alternative functional SaGluCl channel. Reference is made to transgenes and genes for the transgenic animals of the invention. The transgene used in the present invention is a genetic construct containing the gene. By methods known in the art (eg, US Pat. No. 5,612,205, US Pat. No. 5,721,367, US Pat. No. 5,46).
4,764, US Patent No. 5,487,992, US Patent No. 5,627,05.
9 and US Pat. No. 5,631,153), the transgene is integrated into one or more chromosomes in the cells of the animal. Once integrated, the transgene is retained in at least one position within the chromosome of the transgenic animal. Of course, a gene is a nucleotide sequence that encodes a protein such as one of the cDNA clones described herein or a combination thereof. The gene and / or transgene may also include genetic regulatory and / or structural elements known in the art. Target cell types for transgene transfer are embryonic stem cells (ES)
Is. ES cells can be obtained from pre-implantation embryos cultured in vitro,
It is possible to fuse with the embryo (Evans et al., 1981, Nature 29).
2: 154-156; Bradley et al., 1984, Nature 309: 2.
55-258; Gossler et al., 1986, Proc. Natl. Acad.
Sci. USA 83: 9065-9069; and Robertson et al., 1
986 Nature 322: 445-448). The transgene can be efficiently introduced into ES cells by various standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resulting transformed ES cells can then be combined with blastocysts from non-human animals. The introduced ES cells then colonize the embryo and contribute to the germ line of the resulting chimeric animal (Jaenisch, 1988, Science).
e 240: 1468-1474). In addition, wild-type nematodes (C. elegans)
And C. elegans GluC in the GluCl background
nematodes (C. ele) knocked out for one or both of the 1 subunits.
transgenic or knockout invertebrates expressing the SaGluCl2 transgene in a mutant (eg C. elegans).
)) Is within the skill of one in the art. These organisms are Sa
It would be useful for further confirmation of the predominant inhibitory effect of GluCl2 and selection of compounds that modulate this effect.

Pharmaceutically useful compositions containing modulators of SaGluCl can be formulated according to known methods, such as admixing a pharmaceutically acceptable carrier. Specific examples of such carriers and methods of formulation can be found in Remington's Pharmaceutical
al Sciences. In order to form a pharmaceutically acceptable composition suitable for effective administration, such composition comprises the protein, DNA,
It will contain an effective amount of RNA, modified SaGluCl or an agonist or antagonist of SaGluCl, including activators or inhibitors of tyrosine kinases.

The therapeutic or diagnostic compositions of the invention are administered to an individual in an amount sufficient to treat or diagnose the disorder. The effective amount may vary depending on various factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.

The pharmaceutical composition can be administered to an individual by a variety of routes including subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” means a molecule that contains an additional chemical moiety that is not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorbency, etc. of the base molecule. Alternatively, the moiety may reduce undesired side effects of the base molecule or reduce the toxicity of the base molecule. A specific example of such a part is Remington's Pharmaceutical Sc.
It has been described in various publications such as iences.

The compounds identified according to the methods disclosed herein can be used alone at a suitable dose. Alternatively, simultaneous or sequential administration of other substances may be desirable.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing a compound identified in accordance with the present invention as an active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds may be tablets, capsules (including timed release and sustained release formulations, respectively), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups. , As an oral dosage form such as emulsion, or by injection. Similarly, they can be administered as an intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical (with or without occlusion) or intramuscular form, which It is possible to use all forms which are familiar to the person skilled in the art of pharmacy.

It may be advantageous to administer the compounds of the invention in a single daily dose, or to administer a total daily dose in divided doses of 2, 3 or 4 times daily. In addition, the compounds for the present invention may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. You can Of course, in order to be administered in the form of a transdermal delivery system, the administration of the dose will be continuous rather than intermittent throughout the dosing regimen.

In the case of combination therapy with two or more active agents in separate dosage formulations, the active agents may be administered simultaneously or each of them may be administered at different staggered times. .

Dosage regimens using the compounds of the present invention include patient type, species, age, weight, sex and medical condition; severity of the condition to be treated; route of administration; patient renal, hepatic and cardiovascular function. ;, And various factors including the particular compound used. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal and accurate acquisition of drug concentrations within the range that provides efficacy without toxicity requires a kinetics-based program of drug availability to target sites. This includes consideration of drug distribution, equilibrium and elimination.

The following examples serve to illustrate the invention without limiting it.

Example 1 Isolation and Characterization of a DNA Molecule Encoding SaGluCl Ausubel et al. (1992, Short protocols in mo).
regular biology, F.L. M. Ausubel et al., 2nd edition (Joh
n Wiley & Sons)) and Sambrook et al. (1989, Mo.
regular cloning. A laboratory manual.
J. Sambrook, E .; F. Fritsch and T.W. Maniatis,
Second Edition (Cold Spring Harbor Laboratory Pr)
Molecular manipulations were performed according to standard methods well known in the art, such as those described in publications such as ess)). TRI-REAGENT (Molec. Res. Inc.
． ) From a neuron dissected from the ventral ganglion of the grasshopper
NA was isolated. SUPERSCRIPT Preamplification System (Life T
techniques from 200 ng of RNA to first strand cDNA.
Was synthesized. One tenth of the first strand reaction was used for PCR. The degenerate oligos used were C. elegans and Drosophila.
) And a sequence obtained from C. felis GluCl. The oligonucleotides used are as follows.

Forward (Oligo 27F2): GGAT (G / T) CCNGA (C / T) N (C / T) NTT (C / T) TTN
N (A / C) NA (A / C) (C / T) G (SEQ ID NO: 11).

Reverse 1 (Oligo 3B): CNA (A / G) (A / C) A (A / G) NGCNC (A / C) GAANA (C
/ T) (A / G) AA (C / T) G (SEQ ID NO: 12).

Reverse 2 (Oligo 3A): CAN (A / G) CNCCN (A / G) (G / T) CCANAC (A / G) TC
NA (C / T) N (A / G) C (SEQ ID NO: 13).

2 rounds of PC using a combination of 27F2 + 3A followed by 27F2 + 3B
R was done. The cycle was as follows: 1 × (95 ° C. for 120 seconds)
Then 30 × (95 ° C. for 45 seconds, 50 ° C. for 90 seconds and 72 ° C. for 120 seconds), then 1 × (72 ° C. for 120 seconds). Reagent is Life Technology
gy Inc. It is from. The oligo concentration was 5 μM. To identify and prevent PCR cloning of contaminating sequences from GluCl clones already used in the laboratory, one tenth of the PCR reaction products were tested by Southern blot analysis. "TA" Cloning Kit (Invitrogen, In
c. ) Was used to clone a new PCR product of the appropriate size into the PCR2.1 plasmid vector. Sequence analysis (ABI Prism, PE Appl
ied Biosystems), the selected PCR clone insert was radiolabeled and used as a probe with the poly (A) enriched fraction from the RNA to Uni-ZAP vector (Stratagene, Inc. The cDNA library prepared in () was screened. The sequence obtained from the full-length cDNA clone (ABI Prism, PE Applied Biosystems) was analyzed by GCG Inc. Analyzed using the package. Subcloning SaGluCl1 and SaGluCl2 into a mammalian expression vector,
Excision of all inserts from the UniZap pBS plasmid (EcoRI +
(Excision with XhoI) and subsequent TetSplice (Cat. No.
10583-011; Life Technologies / GIBCO BR
L). 1A-1F and 2A-2B show SaGlu.
The deduced nucleotide and amino acid sequences containing the open reading frames of the Cl1 and SaGluCl2 cDNA clones are shown.

Example 2 Functional Expression of GluCl Clones in Xenopus Oocytes Full-length cDNA clones corresponding to selected RT-PCR sequences were templated for synthesis of in vitro transcribed RNA (Ambion Inc.). Used as. pB
full-length c encoding SaGluCl1 in luesscript plasmid
Linearize DNA with appropriate oligonucleotide primers and mMESSAG
EmMACHINE in vitro RNA transcription kit (Ambion)
To synthesize a capped cRNA transcript. Xenopus laevis (Xe
Nopus laevis oocytes were prepared and prepared as described (Ar
ena et al., 1991, Mol. Pharmacol. 40: 368-374; A
rena et al., 1992. Mol. Brain Res. 15: 339-348)
Injection was done by standard methods. Adult female Xenopus (Xenopu)
slaevis) was anesthetized with 0.17% tricaine methanesulfonate, the ovaries were surgically removed, and NaCl adjusted to pH 7.5 with NaOH (OR-2).
82.5, KCl 2, MgCl ₂ 1, CaCl ₂ 1.8, HEPES 5
It was placed in a dish of (mM). 2 in OR-2 containing 0.2% collagenase (Sigma, type 1A), ruptured and opened ovarian lobes, rinsed several times
Shake gently for ~ 5 hours. After removal of about 50% of the follicular layer, stage V and VI oocytes were selected and adjusted to pH 7.5 with NaOH (ND-96) NaCl 86, KCl 2, MgCl ₂ 1, CaCl ₂ 1. 8, HEPE
S 5, Na birubinate 2.5, Theophylline 0.5, Gentamicin 0
． Placed in medium consisting of 1 (mM) for 24-48 hours before injection. In most experiments, 10 ng of cRNA in 50 nl of water without RNase was injected into oocytes. Control oocytes were injected with 50 nl of water. 50 mg before recording
Oocytes were incubated in ND-96 supplemented with / ml gentamicin, 2.5 mM Na pyruvate and 0.5 mM theophylline for 1-5 days. Incubation and collagenase digestion were performed at 18 ° C.

Dagan CA1 Amplifier (Dagan Instruments, Minn
A voltage clamp study was performed with a voltage clamp technique with two microelectrodes (Eapolis, MN). The microelectrode through which the current passes is 0.7 M KCl and 1.7.
M citric acid K ₃ was loaded and the electrogram microelectrodes were loaded with 1.0 M KCl.
The extracellular solution of most experiments was NaCl adjusted to pH 7.5 with NaOH.
96, BaCl ₂ 3.5, MgCl ₂ 0.5, CaCl ₂ 0.1, HEP
It was a saline solution consisting of ES 5 (mM). In some experiments, equimolar substitution of NaCl with the indicated sodium salt of the anion reduced extracellular chloride concentration. The experiment was conducted at 21-24 ° C. Data was obtained using the program Pulse and most analyzes were performed using the companion program Pulsefit (In
strutech Instruments, Great Neck, NY) or Igor Pro (Wavemetrics, Lake Oswego, O)
R). Unless otherwise indicated, data is for a 1 kHz filter (fc,-
3db). FIG. 3 shows the results of an experiment in which clone SaGluCl1 (short form “SC”) was expressed in Xenopus oocytes. The measurements were performed as described in this example by the voltage clamp technique of two microelectrodes and the membrane potential was maintained at 0 mV. Two current records are overlaid. The top bar shows the duration of application of glutamate and ivermectin phosphate. First,
Glutamic acid was applied, which immediately evoked a desensitizing current. Ivermectin phosphate evoked a current of similar amplitude, but the channel remained open for a much longer time. This indicates that expression of this protein reconstitutes a functional ion channel that responds to both glutamate and ivermectin.

Example 3 Functional expression of GluCl clones in mammalian cells Lipofectamine Plus reagent (Life Technology).
ies Inc. ) Is used to prepare SaGluCl1 and Sa as described in Example 1.
PTet-Splice subclone of GluCl2 was CHO Tet-Off
Transfected into cells (Clontech labs, Inc.). G
Stable cell lines are selected by growth in the presence of 418. A single G418 resistant clone is isolated. It has been shown to contain the intact SaGluCl gene. Using immunological techniques such as immunoprecipitation, Western blot and immunofluorescence using antibodies specific for the SaGluCl protein, the SaGluC
Clones containing l cDNA can be analyzed. From a rabbit inoculated with a peptide synthesized from the amino acid sequence deduced from the SaGluCl sequence,
Antibodies can be obtained. Expression is also analyzed using patch clamp electrophysiological techniques, anion flux assays and ³ H-ivermectin and ³ H-glutamate binding assays.

Cells that stably or transiently express SaGluCl are used to test for expression of avermectin, glutamate-sensitive chloride channels and for ligand binding activity. Using these cells, other compounds are identified and studied for their ability to regulate, inhibit or activate avermectin, glutamate sensitive chloride channels and compete for binding to radioactive avermectin, a glutamate derivative. These cells are used to identify and study other compounds that regulate SaGluCl activity by anion flux assay.

A cassette containing the SaGluCl cDNA in a positive orientation relative to the promoter is ligated into the appropriate restriction sites 3'to the promoter and identified by restriction site mapping and / or sequencing. These cDNA expression vectors are transformed into host cells, eg CHO cells, COS-, by standard methods including, but not limited to, electroporation or chemical methods (cationic liposomes, DEAE dextran, calcium phosphate). 7 (ATCC #
CRL1651) and CV-1 tat [Sackevitz et al., Science.
238: 1575 (1987)], 293, L (ATCC # CRL636.
2)]. Transfected cells and cell culture supernatants can be collected and analyzed for SaGluCl expression.

All vectors used for mammalian transient expression can be used to establish stable cell lines expressing SaGluCl. The unmodified SaGluCl cDNA construct cloned into the expression vector is expected to direct the host cell to produce SaGluCl protein. Also, by ligating the SaGluCl GluCl cDNA construct to DNA encoding the signal sequence of a secreted protein, SaGluCl is expressed extracellularly as a secreted protein. CV-1-P [Sackevitz et al., S.
science 238: 1575 (1987)], tk-L [Wigler et al.
Cell 11: 223 (1977)], NS / 0 and dHFr-CHO [K.
aufman and Sharp, J .; Mol. Biol. 159: 601, (1
982)], but is not limited thereto.

G418, aminoglycoside phosphotransferase; hygromycin,
Co-transfection of drug selection plasmids, including (but not limited to) hygromycin-B phosphotransferase; APRT, xanthine-guanine phosphoribosyl-transferase, with any vector containing SaGluCl GluCl is stable. It will allow the selection of transfected clones. The assay described herein allows SaGlu
Quantify the level of Cl GluCl.

In addition, for the production of mammalian cell clones that synthesize the highest level of SaGluCl possible, SaGluC in a vector containing an amplifiable drug resistance marker.
l ligate the cDNA construct. After introducing these constructs into cells, clones containing the plasmid are selected with the appropriate agent, and isolation of overexpressing clones with high copy number plasmid is selected with increasing amounts of the agent. By.

Expression of recombinant SaGluCl1 and / or SaGluCl2 is achieved by transfection of full-length SaGluCl cDNA into mammalian host cells as described herein. Functional expression of SaGluCl1 in CHO cells is shown in FIGS. 4A and 4B. This is because this subunit is ivermectin (IV
M, FIG. 4A) and nodular sporic acid (NA, FIG. 4B) are shown to be able to assemble into homomultimeric channels.

Example 4 SaGlu into Baculovirus Expression Vector for Expression in Insect Cells
Cloning of Cl GluCl cDNA The baculovirus vector derived from the AcNPV genome was cloned into Sf of insect cells.
Designed to give high level expression of cDNA within the 9 line (ATCC CRL # 1711). Recombinant baculovirus expressing SaGluCl cDNA was prepared according to the following standard method (In Vitrogen Maxbac Manual).
l). pAC360 and BlueBac vectors (In Vitr
The SaGluCl cDNA construct is ligated into the polyhedrin gene in various baculovirus transfer vectors, including The baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, 1990, Nuc. Ac
id. Res. 18: 5667] and Sf9 cells are co-transfected, and then homologous recombination is performed to obtain a recombinant baculovirus. Recombinant pAC360 virus was identified by the absence of inclusion bodies in infected cells,
The eBac virus is identified based on the expression of b-galactosidase (Sum
mers, M .; D. And Smith, G .; E. , Texas Agricul
pure Exp. Station Bulletin No. 1555). After plaque purification, SaGluCl expression is measured by the assay described herein.

SaGluCl The cDNA encoding the entire open reading frame of GluCl is inserted into the BamHI site of pBlueBacII. A positive orientation construct was identified by sequence analysis and used to determine linear AcNPV relaxed DNA.
Sf9 cells in the presence of

Authentic active SaGluCl is found in the cytoplasm of infected cells. Active SaGluCl is extracted from infected cells by hypotonic or detergent lysis.

Example 5 Cloning of SaGluCl GluCl cDNA into a Yeast Expression Vector After inserting the optimal SaGluCl cDNA cistron into an expression vector designed to direct intracellular or extracellular expression of a heterologous protein, the yeast Saccharomyces Recombinant SaGluCl in S. cerevisiae
To produce. In the case of intracellular expression, a vector such as EmBLyex4 is used as the S
ligated to aGluCl cistron [Rinas et al., 1990, Biotech
No. 8: 543-545; Horowitz B et al., 1989, J. Am.
Biol. Chem. 265: 4189-4192]. In the case of extracellular expression,
Secretion signals (yeast or mammalian peptides) are transferred to the SaGluCl protein N
A yeast expression vector fused to the H ₂ terminus [Jacobson, 1989, Gene.
85: 511-516; Riett and Bellon, 1989, Bioc.
hem. 28: 2941-2949] into the SaGluCl GluCl cistron.

These vectors include pAVE1-6 [Step, 1990, Biotechnology 8], which fuses the human serum albumin signal to the expressed cDNA.
: 42-46], and a vector pL8PL [Yamamoto, Biochem. 28: 2728-27
32], but is not limited thereto. In addition, the vector pVE
P [Ecker, 1989, J. Biol. Chem. 264: 7715-77
19, Sabin, 1989 Biotechnology 7: 705-70.
9, McDonnell, 1989, Mol. Cell Biol. 9: 551
7-5523 (1989)], SaGluCl is expressed in yeast as a fusion protein coupled to ubiquitin. The level of SaGluCl expressed is measured by the assay described herein.

Example 6 Purification of Recombinant SaGluCl CluCl Recombinantly produced SaGluCl can be purified by antibody affinity chromatography. The SaGluCl GluCl antibody affinity column was loaded onto the SaGlu which is a gel support pre-activated with N-hydroxysuccinimide ester so that the SaGluCl GluCl antibody forms a covalent bond with the agarose gel bead support.
Make by adding Cl GluCl antibody. The antibody is then attached to the gel via an amide bond with a spacer arm. The remaining activated ester is then quenched with 1M ethanolamine HCl (pH 8). The column is washed with water followed by 0.23M glycine HCl (pH 2.6) to remove unconjugated antibody or foreign protein. The column was then loaded with phosphate buffered saline (pH 7.
3) and equilibrated in a suitable membrane solubilizer (eg detergent) to solubilize Sa
Cell culture supernatant or cell extract containing GluCl is slowly passed through the column. Then until the optical density (A ₂₈₀ ) drops to the background,
The column is washed with phosphate buffered saline and detergent, then the protein is eluted with 0.23M glycine-HCl (pH 2.6) and detergent. The purified SaGluCl protein is then dialyzed against phosphate buffered saline.

[Sequence list]

[Brief description of drawings]

FIG. 1A is the respective grasshopper GluC set forth in SEQ ID NOs: 1, 3, 5, 7 and 9.
The nucleotide sequences of the DNA molecules encoding the I proteins are compared.

FIG. 1B Respective grass hopper GluC set forth in SEQ ID NOs: 1, 3, 5, 7 and 9.
The nucleotide sequences of the DNA molecules encoding the I proteins are compared.

FIG. 1C: The respective grasshopper GluC set forth in SEQ ID NOs: 1, 3, 5, 7 and 9.
The nucleotide sequences of the DNA molecules encoding the I proteins are compared.

FIG. 1D is the respective grasshopper GluC set forth in SEQ ID NOs: 1, 3, 5, 7 and 9.
The nucleotide sequences of the DNA molecules encoding the I proteins are compared.

FIG. IE: Respective grass hopper GluC set forth in SEQ ID NOs: 1, 3, 5, 7 and 9.
The nucleotide sequences of the DNA molecules encoding the I proteins are compared.

FIG. 1F Respective grass hopper GluC set forth in SEQ ID NOs: 1, 3, 5, 7 and 9.
The nucleotide sequences of the DNA molecules encoding the I proteins are compared.

FIG. 2A shows the amino acid sequences of grasshopper GluCl proteins set forth in SEQ ID NOs: 2, 4, 6, 8 and 10.

FIG. 2B shows the amino acid sequences of grasshopper GluCl proteins set forth in SEQ ID NOs: 2, 4, 6, 8 and 10.

FIG. 3. Clone SaGluCl1 (short form “SC”) was cloned in Xenopus.
s) Shows the results of experiments expressed in oocytes.

FIG. 4A shows SaGluCl1 (short form “SC”) expressed in CHO cells, assembled into homomultimers and activated by ivermectin.

FIG. 4B shows SaGluCl1 (short form “SC”) activated by nodulasporic acid.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12Q 1/02 4H045 C12P 21/02 1/68 Z C12Q 1/02 G01N 33/483 F 1 / 68 C12N 15/00 ZNAA G01N 27/416 5/00 A 33/483 G01N 27/46 336G (72) Inventor Wormky, Jeffrey Davrilleu, USA, New Jersey 07065-0907, Lowway, East Linkarn・ Avenyu ・ 126 F term (reference) 2G045 AA34 AA35 BB20 BB50 BB51 CB01 CB17 DA13 DA36 FB02 FB05 GC18 4B024 AA01 AA11 CA04 HA01 4B063 QA18 QR80 QS12 QS36 QX05 4B064 AG01 CA19B20 CA30B24A12 BA01 4A0 A4 BA01 4A0 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A3 A4 EA06 EA50 FA74

Claims (57)

[Claims] 1. A S. americana G.
A purified DNA molecule encoding the luCl1 channel protein. 2. embedded image Cystocerca americana (S.
americana) The purified DNA molecule of claim 1, which encodes a GluCl1 channel protein. 3. An expression vector for expressing a S. americana GluCl1 channel protein in a recombinant host cell, which comprises the DNA molecule of claim 1. 4. A host cell containing the expression vector of claim 3 and expressing a recombinant S. americana GluCl1 channel protein. 5. Cyst cerca americana (S. am) in a recombinant host cell.
ericana) a method for expressing a GluCl1 channel protein, comprising: (a) transfecting the expression vector of claim 3 into a suitable host cell; (b) the Cystocerca americana (S) from the expression vector. .Ameri
cana) under conditions that allow expression of the GluCl channel protein, step (
A method comprising culturing the host cell of a). 6. An expression vector for expressing a S. americana GluCl1 channel protein in a recombinant host cell, which comprises the DNA molecule of claim 2. 7. A host cell containing the expression vector of claim 6 and expressing a recombinant S. americana GluCl1 channel protein. 8. A cystocerca americana (S. am) in a recombinant host cell.
ericana) a method for expressing a GluCl-1 channel protein, comprising: (a) transfecting the expression vector of claim 6 into a suitable host cell; (b) said Cytocelka americana from said expression vector. (S. ameri
cana) under conditions that allow expression of the GluCl channel protein, step (
A method comprising culturing the host cell of a). 9. embedded image Cytoselka americana (S consisting of an amino acid sequence selected from the group consisting of
． americana) A purified DNA molecule encoding a GluCl1 channel protein. 10. An expression vector for expressing a S. americana GluCl1 channel protein in a recombinant host cell, which comprises the DNA molecule of claim 9. 11. A host cell expressing the recombinant S. americana GluCl1 channel protein containing the expression vector of claim 10. 12. Cyst cerca americana (S. a.
mericana) A method for expressing a GluCl-1 channel protein, comprising: (a) transfecting the expression vector of claim 10 into a suitable host cell; (b) the Cystcella americana from the expression vector. (S. ameri
cana) under conditions that allow expression of the GluCl channel protein, step (
A method comprising culturing the host cell of a). 13. Amino acid sequence: A purified DNA molecule encoding a S. americana GluCl2 channel protein comprising 14. An expression vector for expressing a S. americana GluCl2 channel protein in a recombinant host cell, which comprises the DNA molecule of claim 13. 15. A host cell expressing a recombinant S. americana GluCl2 channel protein containing the expression vector of claim 14. 16. Cyst cerca americana (S.a) in a recombinant host cell.
mericana) A method for expressing a GluCl2 channel protein, comprising: (a) transfecting the expression vector of claim 14 into a suitable host cell; (b) the Cystocerca americana (S) from the expression vector. .Ameri
cana) under conditions that allow expression of the GluCl channel protein, step (
A method comprising culturing the host cell of a). 17. Amino acid sequence: Sist americana GluCl2 consisting of
A purified DNA molecule encoding a channel protein. 18. An expression vector for expressing a S. americana GluCl2 channel protein in a recombinant host cell, which comprises the DNA molecule of claim 17. 19. A host cell that expresses a recombinant S. americana GluCl2 channel protein containing the expression vector of claim 18. 20. Cytoselka americana (S. a.
mericana) A method for expressing a GluCl2 channel protein, comprising: (a) transfecting the expression vector of claim 18 into a suitable host cell; (b) the Cytocelka americana (S) from the expression vector. .Ameri
cana) under conditions that allow expression of the GluCl channel protein, step (
A method comprising culturing the host cell of a). 21. A recombinant S. americana GluCl channel protein comprising a nucleotide sequence selected from the group of nucleotide sequences consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. A purified DNA molecule that encodes. 22. Recombinant S. americana GluC in a recombinant host cell comprising the DNA molecule of claim 21.
An expression vector for expressing an l1 channel protein. 23. A host cell expressing a recombinant S. americana GluCl1 channel protein containing the expression vector of claim 22. 24. In a recombinant host cell, recombinant Cystselka americana (
S. americana) A method for expressing a GluCl1 channel protein, comprising: (a) transfecting the expression vector of claim 22 into a suitable host cell; (b) the recombinant Cystoluca americana from the expression vector. (S. Am
ericana) a method comprising culturing the host cell of step (a) under conditions that allow expression of the GluCl1 channel protein. 25. A purified encoding a recombinant S. americana GluCl1 channel protein comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. DNA molecule. 26. Recombinant S. americana GluC in a recombinant host cell, comprising a DNA molecule according to claim 25.
An expression vector for expressing an l1 channel protein. 27. A host cell expressing a recombinant S. americana GluCl1 channel protein containing the expression vector of claim 26. 28. Recombinant Cystocerca americana in a recombinant host cell
S. americana) A method for expressing a GluCl1 channel protein, comprising: (a) transfecting the expression vector of claim 26 into a suitable host cell; (b) the recombinant Cystoluca americana from the expression vector. (S. Am
ericana) a method comprising culturing the host cell of step (a) under conditions that allow expression of the GluCl1 channel protein. 29. A purified DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 9 and encoding a recombinant S. americana GluCl2 channel protein. 30. Recombinant S. americana GluC in a recombinant host cell comprising the DNA molecule of claim 29.
An expression vector for expressing the 12 channel protein. 31. A host cell expressing a recombinant S. americana GluCl2 channel protein containing the expression vector of claim 30. 32. Recombinant Cystocerca americana in a recombinant host cell
S. americana) A method for expressing a GluCl2 channel protein, comprising: (a) transfecting the expression vector of claim 30 into a suitable host cell; (b) the recombinant Cystocerca americana from the expression vector. (S. Am
ericana) A method comprising culturing the host cell of step (a) under conditions that allow expression of the GluCl2 channel protein. 33. A purified DNA molecule encoding a recombinant S. americana GluCl2 channel protein consisting of the nucleotide sequence set forth in SEQ ID NO: 9. 34. Recombinant S. americana GluC in a recombinant host cell comprising the DNA molecule of claim 33.
An expression vector for expressing the 12 channel protein. 35. A host cell expressing the recombinant S. americana GluCl2 channel protein containing the expression vector of claim 34. 36. Recombinant Cystocerca americana in a recombinant host cell
S. americana) A method for expressing a GluCl2 channel protein, comprising: (a) transfecting the expression vector of claim 34 into a suitable host cell, (b) the recombinant Cytoselka americana from the expression vector. (S.am
ericana) A method comprising culturing the host cell of step (a) under conditions that allow expression of the GluCl2 channel protein. 37. Another S. americana (S. americana)
a) Cystselka americana (S. ame) substantially free of protein
ricana) GluClu-1 channel protein. 38. The S. americana GluCl1 channel protein of claim 37, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. 39. The S. americana of claim 38, which is the product of a DNA expression vector contained in a recombinant host cell.
a) GluCl1 channel protein. 40. A substantially pure membrane preparation comprising a S. americana GluCl1 channel protein purified from the recombinant host cell of claim 39. 41. Cystocerca americana (S. cerevisiae) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
americana) GluCl1 channel protein. 42. The S. americana of claim 41, which is the product of a DNA expression vector contained in a recombinant host cell.
a) GluCl1 channel protein. 43. A substantially pure membrane preparation comprising a S. americana GluCl1 channel protein purified from the recombinant host cell of claim 42. 44. Another S. americana
a) Cystselka americana (S. ame) substantially free of protein
ricana) GluClu-2 channel protein. 45. The S. americana GluCl2 channel protein of claim 44, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. 46. The S. americana of claim 45, which is the product of a DNA expression vector contained in a recombinant host cell.
a) GluCl2 channel protein. 47. A substantially pure membrane preparation comprising a S. americana GluCl2 channel protein purified from the recombinant host cell of claim 46. 48. Cystocerca americana (S. cerevisiae) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
americana) GluCl2 channel protein. 49. The S. americana of claim 41, which is the product of a DNA expression vector contained in a recombinant host cell.
a) GluCl2 channel protein. 50. A substantially pure membrane preparation comprising a S. americana GluCl2 channel protein purified from the recombinant host cell of claim 42. 51. A method for identifying a compound that modulates the activity of a glutamate-gated channel protein, comprising: a) a population of nucleic acid molecules, at least a portion of which is Cystcelka americana (S
． americana) encodes the GluCl1 channel protein,
The expression of said nucleic acid molecule part provides an active glutamate-gated channel) is injected into the host cell solution, b) the test compound is added to said solution, and c) the host at a holding potential more positive than the reversal potential of chloride A method comprising measuring cell membrane current. 52. The nucleic acid molecule is complementary DNA, poly A ⁺ messenger R.
52. The method of claim 51, selected from the group consisting of NA and complementary RNA. 53. The S. americana
) GluCl1 channel proteins are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6
52. The method of claim 51 selected from the group consisting of and SEQ ID NO: 8. 54. A method of identifying a compound that modulates the activity of a glutamate-gated channel protein, comprising: a) a population of nucleic acid molecules, at least a portion of which is Cystcerca americana (S).
． americana) GluCl1 channel protein and S. americana GluCl2 protein, the expression of which part of the nucleic acid molecule confers an active glutamate-gated channel) into the host cell solution, and b) the test Adding a compound to the solution, and c) measuring the host cell membrane current at a holding potential more positive than the reversal potential of chloride. 55. The nucleic acid molecule is complementary DNA, poly A ⁺ messenger R.
55. The method of claim 54, selected from the group consisting of NA and complementary RNA. 56. The S. americana
55.) The method of claim 54, wherein the GluCl2 channel protein comprises SEQ ID NO: 10. 57. The S. americana
) GluCl1 channel proteins are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6
57. The method of claim 56, selected from the group consisting of and SEQ ID NO: 8.